Methods for treating progeroid laminopathies using oligonucleotide analogues targeting human lmna

ABSTRACT

Provided are methods of treatment in subjects having progeroid diseases and related conditions which rely upon LMNA-targeted antisense oligonucleotides for reducing expression of one or more aberrantly spliced LMNA mRNA isoforms that encode progerin.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 120178_49401_SEQUENCE_LISTING.txt. The text fileis about 14 KB, was created on Dec. 6, 2012, and is being submittedelectronically via EFS web.

BACKGROUND

Technical Field

The present invention relates generally to methods of treatment forprogeroid laminopathies including Hutchinson-Gilford progeria syndromeusing human lamin A targeted antisense compounds and relatedcompositions.

Description of the Related Art

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disordercharacterized by premature arteriosclerosis and degeneration of vascularsmooth muscle cells (SMCs). HGPS manifests itself most notably asaccelerated, premature aging in affected children. Children with HGPShave progressive symptoms such as growth retardation, alopecia, loss ofsubcutaneous fat, and bone abnormalities. Average lifespan is 12 yearswith the most common cause of death being myocardial infarction orstroke.

Most HGPS cases are caused by a single-point mutation in the lamin A(LMNA) gene, resulting in the generation of progerin, a truncatedsplicing mutant of lamin A. The single-point mutation is a de novosilent substitution (1824C>T, Gly608Gly) in exon 11 of the lamin A(LMNA) gene. The substitution activates a cryptic splice donor site,which leads to the production of a dominant negative mutant lamin Aprotein with an internal deletion of 50 amino acids. The mutant protein,named progerin, accumulates on the nuclear membrane, causingcharacteristic nuclear blebbing ((Scaffidi and Misteli 2005; Cao, Blairet al. 2011)).

It is known that aberrant splicing can be corrected usingphosphorodiamidate morpholino oligonucleotides (PMOs), or morespecifically, splice-switching oligonucleotides (SSOs). SSOs blockaberrant splicing sites by hybridizing at or near the sites therebypreventing recognition by the cellular splicing machinery. PreferredSSOs are resistant to nucleases and the resulting double-strandedstructure eliminates the possibility of RNA cleavage by RNase H. SSOshave been shown to effectively repair the splicing pattern both in vitroand in vivo for thalassemia and Duchenne muscular dystrophy. (Kinali,Arechavala-Gomeza et al. 2009; Svasti, Suwanmanee et al. 2009). Theaberrant splicing of LMNA associated with HGPS has been shown to bereduced by correction of the aberrant splicing event using modifiedantisense oligonucleotides targeted to the activated cryptic splice siteboth in cell culture (Scaffidi and Misteli 2005) and in a relevantanimal model (Osorio, Navarro et al. 2011).

Given the role of LMNA in HGPS, improved oligonucleotide-based methodsof treatment targeting LMNA pre-mRNA are needed.

BRIEF SUMMARY

Embodiments of the present invention relate generally to methods fortreating a progeroid disease or related condition in a subject in needthereof comprising administering to the subject an antisenseoligonucleotide, wherein the oligonucleotide modulates aberrant splicingof a human LMNA pre-mRNA, the oligonucleotide being composed ofmorpholino subunits and phosphorus-containing intersubunit linkagesjoining a morpholino nitrogen of one subunit to a 5′-exocyclic carbon ofan adjacent subunit, and:

-   -   (i) having a substantially uncharged, nuclease resistant        backbone;    -   (ii) capable of uptake by mammalian host cells;    -   (iii) containing between about 12-40 nucleotide bases;    -   (iv) having a targeting sequence of at least about 12 contiguous        subunits complementary to exon 10, intron 10, exon 11, or        combinations thereof, of a human LMNA pre-mRNA; and

The methods of the invention can be used for the treatment of diseases,such as diseases or conditions associated with human LMNA. In certainembodiments, the methods are used in the treatment of progeroid diseasesor related conditions, such as a progeroid laminopathy (such as HGPS),an age-related condition, or a cardiovascular disease (such asatherosclerosis).

In more specific embodiments of the methods, the targeting sequence ofthe oligonucleotide is complementary to bases upstream of the exon 11cryptic splice site of a human LMNA pre-mRNA. In other specificembodiments, the targeting sequence is complementary to bases downstreamof the exon 11 cryptic splice site of a human LMNA pre-mRNA. In stillother specific embodiments, the targeting sequence does not overlap theexon 11 cryptic splice site of the human LMNA pre-mRNA.

In additional embodiments of the methods herein, the progeroid diseaseis associated with the 1824C>T mutation within the exon 11 crypticsplice site of the human LMNA pre-mRNA. In related embodiments, thetargeting sequence in the oligonucleotide used in the methods of theinvention does not overlap with the 1824C>T mutation.

In another specific embodiment, the 3′-most base of the targetingsequence is complementary to a base in LMNA exon 11 that is about 1-30bases downstream of the exon 11 cryptic splice site of a human LMNApre-mRNA.

In yet another specific embodiment, the 3′-most base of the targetingsequence is complementary to a base in LMNA exon 11 that is about 10-40bases upstream of the exon 11 cryptic splice site of a human LMNApre-mRNA.

In still another specific embodiment, the 3′-most base of the targetingsequence is complementary to a base in LMNA intron 10 that is about 1-60bases upstream of LMNA exon 11.

In another specific embodiment, the 3′-most base of the targetingsequence is complementary to a base in LMNA exon 10 that is about 1-30bases upstream of LMNA intron 10.

In still another specific embodiment, the targeting sequence iscomplementary to a region that overlaps the splice junction of splicedonor (SD) or splice acceptor (SA) sites of exons 10 and 11 of LMNApre-mRNA, and is complementary to a portion of an exonic region and aportion of an intronic region of the pre-processed mRNA.

The targeting sequence of the oligonucleotide used in the methods, ineven more specific embodiments of the invention, is complementary to atleast 12 contiguous bases of any one of SEQ ID NOs: 3-34, or is at least90% identical to any one of SEQ ID NOs: 3-34, or comprises any one ofSEQ ID NOs: 3-34, or consists of any one of SEQ ID NOs: 3-34.

The targeting sequence of the oligonucleotide used in any of themethods, in even more specific embodiments of the invention, iscomplementary to at least 12 contiguous bases of any one of SEQ ID NOs:3-7 or 14-16, or is at least 90% identical to any one of SEQ ID NOs: 3-7or 14-16, or comprises any one of SEQ ID NOs: 3-7 or 14-16, or consistsof any one of SEQ ID NOs: 3-7 or 14-16.

The targeting sequence of the oligonucleotide used in any of themethods, in even more specific embodiments of the invention, iscomplementary to at least 12 contiguous bases of SEQ ID NO: 4, or is atleast 90% identical to SEQ ID NO: 4, or comprises SEQ ID NO: 4, orconsists of SEQ ID NO: 4.

The targeting sequence of the oligonucleotide used in any of themethods, in even more specific embodiments of the invention, iscomplementary to at least 12 contiguous bases of SEQ ID NO: 11, or is atleast 90% identical to SEQ ID NO: 11, or comprises SEQ ID NO: 11, orconsists of SEQ ID NO: 11.

In other embodiments, the oligonucleotide used in the methods is aphosphorodiamidate morpholino oligonucleotide (PMO), or a PMO comprisingone or more piperazine-containing intersubunit linkages (PMOplus), or aPMO-X oligonucleotide.

Exemplary morpholino subunits, according to certain methods of theinvention, are joined by phosphorodiamidate linkages, in accordance withthe following structure:

wherein Z is S or O,

X=NR¹R² or OR⁶,

Y=O or NR⁷,

and each said linkage is selected from:

(a) uncharged linkage (a), wherein each of R¹, R², R⁶, and R⁷ isindependently selected from hydrogen and lower alkyl; (b1) cationiclinkage (b1), wherein X=NR¹R² and Y=O, and NR¹R² represents an optionalsubstituted piperazino group, such that R¹R²=—CHRCHRN(R³)(R⁴)CHRCHR—,wherein

each R⁴ is H, CH₃ or null, and

R³ is selected from H, lower alkyl, C(═NH)NH₂, Z-L-NHC(═NH)NH₂, and

[C(O)CHR′NH]_(m)H, wherein where Z is carbonyl (C(O)) or a direct bond,L is an optional linker up to 18 atoms in length having bonds selectedfrom alkyl, alkoxy, and alkylamino, R′ is a side chain of a naturallyoccurring amino acid or a one- or two-carbon homolog thereof, and m is 1to 6;

(b2) cationic linkage (b2), wherein X=NR¹R² and Y=O, R¹=H or CH₃, andR²=LNR³R⁴R⁵, wherein L, R³, and R⁴ are defined as above, and R⁵ is H,lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), wherein Y=NR⁷ and X=OR⁶, and R7=LNR³R⁴R⁵.wherein L, R³, and R⁴ and R⁵ are defined as above, and R⁶ is H or loweralkyl; and at least one said linkage is selected from cationic linkages(b1), (b2), and (b3).

In a more specific embodiment of the structure above, each of R¹ and R²,in linkages of type (a), is methyl.

In another specific embodiment of the structure above, at least onelinkage is of type (b1), where each R is H, R⁴ is H, CH₃, or an electronpair, and R³ is selected from H, CH₃, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂.

In another specific embodiment of the structure above, at least onelinkage is of type (b1), where each R is H, R⁴ is an electron pair, andR³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂.

In still another specific embodiment of the structure above, at leastone linkage is of type (b1), where each R is H, R⁴ is an electron pair,and R³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂.

In yet another specific embodiment of the structure above, R³ isC(O)-L-NHC(NH)NH₂, and L is a hydrocarbon having the structure—(CH₂)_(n)—, where n is 1 to 12.

In another specific embodiment of the structure above, at least onelinkage is of type (b1), where each R is H, and each of R³ and R⁴ isindependently H or CH₃.

In other embodiments of the invention, the antisense oligonucleotideused in the methods of the invention is covalently attached to acell-penetrating peptide, such as an arginine-rich peptide. In a morespecific embodiment, the arginine-rich peptide is attached at itsC-terminus to the 5′ end of the oligonucleotide through a one- ortwo-amino acid linker. Alternatively, in another embodiment, the peptideis attached at its C-terminus to the 3′ end of the oligonucleotide usedin the methods of the invention through a one- or two-amino acid linker.In a preferred embodiment the cell-penetrating peptide is linked to theoligonucleotide through a glycine amino acid.

In additional embodiments of the methods of the invention, there isprovided an oligonucleotide comprising a backbone, the backbonecomprising a sequence of morpholino ring structures joined byintersubunit linkages, the intersubunit linkages joining a 3′-end of onemorpholino ring structure to a 5′-end of an adjacent morpholino ringstructure, wherein each morpholino ring structure is bound to abase-pairing moiety, such that the oligonucleotide can bind in asequence-specific manner to a target nucleic acid, comprising atargeting sequence that is complementary to at least 12 bases of asequence set forth in any one of SEQ ID NO: 1-34, or which comprises anyone or more of SEQ ID NOS: 3-34, wherein the intersubunit linkages havethe following general structure (I):

or a salt or isomer thereof, and wherein each of the intersubunitlinkages (I) are independently linkage (A) or linkage (B):

wherein for linkage (A):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³ or;

Y is, at each occurrence, independently O or —NR²,

R¹ is, at each occurrence, independently hydrogen or methyl;

R² is, at each occurrence, independently hydrogen or —LNR⁴R⁵R⁷;

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁴ is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH₂,—Z-L-NHC(═NH)NH₂ or —[C(O)CHR′NH]_(m)H, where Z is carbonyl (C(O)) or adirect bond, R′ is a side chain of a naturally occurring amino acid or aone- or two-carbon homolog thereof, and m is 1 to 6;

R⁵ is, at each occurrence, independently hydrogen, methyl or an electronpair;

R⁶ is, at each occurrence, independently hydrogen or methyl;

R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or C₁-C₆alkoxyalkyl;

L is an optional linker up to 18 atoms in length comprising alkyl,alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and

Y is, at each occurrence, independently O or —NR¹⁰,

R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂ alkyl;

R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂aralkyl or aryl;

R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl or-LNR⁴R⁵R⁷;

wherein R⁸ and R⁹ may join to form a 5-18 membered mono or bicyclicheterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7 memberedheterocycle, and wherein when X is 4-piparazino, X has the followingstructure (III):

wherein:

R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl; and

R is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl; and

R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂ alkyl,C₁-C₁₂ aminoalkyl, —NH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵, C₁-C₁₂ alkylcarbonyl,oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl,amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl; and wherein at least one ofthe intersubunit linkages is linkage (B).

In some embodiments using the structure above, at least 5% of theintersubunit linkages are linkage (B). In a related embodiment, 10% to50% of the intersubunit linkages are linkages (B). In another relatedembodiment, each linkage (B) has the same structure at each occurrence.In yet another specific embodiment of the structure above, each Y andeach W are O.

Still other embodiments of the methods of the invention provide anantisense oligonucleotide comprising a targeting sequence that iscomplementary to one or more bases of exon 10 or exon 11 in the humanLMNA gene and that contains at least 12 contiguous bases complementaryto a sequence set forth in any one of SEQ ID NOs: 3-34.

Further embodiments of the methods of the invention provide an antisenseoligonucleotide comprising a backbone, the backbone comprising asequence of morpholino ring structures joined by intersubunit linkagesof type (A), (B), or combinations thereof, wherein each morpholino ringstructure supports a base-pairing moiety, such that the oligonucleotidecompound can bind in a sequence-specific manner to a target nucleicacid, comprising a target sequence that is complementary to at least 12bases of any one of SEQ ID NOs: 1-34, or which comprises any one or moreof SEQ ID NOs: 3-34, and wherein the oligonucleotide comprises a 3′terminus, a 5′ terminus and has the following structure (XVII):

or a salt or isomer thereof, and

wherein for linkage (A):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³ or;

Y is, at each occurrence, independently 0 or —NR²,

R¹ is, at each occurrence, independently hydrogen or methyl;

R² is, at each occurrence, independently hydrogen or -LNR⁴R⁵R⁷;

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁴ is, at each occurrence, independently hydrogen, C₁-C₆ alkyl,—C(═NH)NH₂, —Z-L-NHC(═NH)NH₂ or —[C(O)CHR′NH]_(m)H, where Z is carbonyl(C(O)) or a direct bond, R′ is a side chain of a naturally occurringamino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

R⁵ is, at each occurrence, independently hydrogen, methyl or an electronpair;

R⁶ is, at each occurrence, independently hydrogen or methyl;

R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or C₁-C₆alkoxyalkyl;

L is an optional linker up to 18 atoms in length comprising alkyl,alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and

Y is, at each occurrence, independently O or —NR¹⁰,

R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂ alkyl;

R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂aralkyl or aryl;

R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl or-LNR⁴R⁵R⁷;

wherein R⁸ and R⁹ may join to form a 5-18 membered mono or bicyclicheterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7 memberedheterocycle, and wherein when X is 4-piparazino, X has the followingstructure (III):

wherein:

R¹⁰ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl; and

R¹¹ is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl;

R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂ alkyl,C₁-C₁₂ aminoalkyl, —NH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵, C₁-C₁₂ alkylcarbonyl,—CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl,amidinylalkylcarbonyl, guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl; and

R¹⁷ is, at each occurrence, independently absent, hydrogen or C₁-C₆alkyl;

R¹⁸ and R¹⁹ are, at each occurrence, independently absent, hydrogen, acell-penetrating peptide, a natural or non-natural amino acid, C₂-C₃₀alkylcarbonyl, —C(═O)OR²¹ or R²⁰;

R²⁰ is, at each occurrence, independently guanidinyl, heterocyclyl,C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl, C₇-C₃₀ aralkyl, C₃-C₃₀alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈ cycloalkylalkylcarbonyl,C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl,C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀aralkyloxycarbonyl, or —P(═O)(R²²)₂;

R²¹ is C₁-C₃₀ alkyl comprising one or more oxygen or hydroxyl moietiesor combinations thereof;

each R²² is independently C⁶-C¹² aryloxy;

B is a base-pairing moiety;

L¹ is an optional linker up to 18 atoms in length comprising bondsselected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester,carbonyl, carbamate, phosphorodiamidate, phosphoroamidate,phosphorothioate, piperazine and phosphodiester;

x is an integer of 0 or greater; and

wherein at least one of R¹⁸ or R¹⁹ is R²⁰ and provided that both of R¹⁷and R¹⁸ are not absent.

The present invention also provides, in other embodiments, methods fortreating progeroid laminopathies by administering a pharmaceuticalcomposition comprising an antisense oligonucleotide as described hereinand a pharmaceutically acceptable excipient.

In another illustrative embodiment, there is provided a method fortreating progeroid laminopathies in a subject in need thereof comprisingadministering to the subject an antisense oligonucleotide orpharmaceutical composition, as described herein, wherein theoligonucleotide inhibits expression of mutant LMNA protein mRNA bymodulating splicing of LMNA pre-mRNA.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures, compounds and/or compositions, and are eachhereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows an exemplary morpholino oligomer structure with aphosphorodiamidate linkage;

FIG. 1B shows a morpholino oligomer as in FIG. 1A, but where thebackbone linkages contain one positively charged group in the form of a(piperazino) phosphorodiamidate linkage;

FIG. 1C shows a conjugate of an arginine-rich peptide and an antisenseoligomer, in accordance with one embodiment of the invention;

FIGS. 1D-G show the repeating subunit segment of exemplary morpholinooligonucleotides, designated D through G.

FIG. 2 shows results for immunofluorescence screening of progerin andlamin A/C.

FIG. 3 shows results for Western analysis of lamin A and progerin.

FIG. 4 shows results for RT-qPCR analysis of lamin A and progerin.

FIG. 5 shows results for Western analysis of lamin A, lamin C andprogerin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for treating progeroidlaminopathies in a subject in need thereof by administering to thesubject an antisense oligonucleotide as described herein, or apharmaceutical composition containing the same, wherein theoligonucleotide inhibits expression of mutant LMNA protein mRNA bymodulating splicing of LMNA pre-mRNA. In certain aspects, for example,these and related methods can be applied to treating progeroidlaminopathies in clinical settings where progerin expression isassociated with a disease such as HGPS. These and related embodimentscan also be combined with methods of treating or reducing progeriodlaminopathies, by concurrently or sequentially carrying out the methodsof the invention with the other treatment.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. While perfect complementarity is oftendesired, some embodiments can include one or more but preferably 6, 5,4, 3, 2, or 1 mismatches with respect to the target RNA. Variations atany location within the oligomer are included. In certain embodiments,variations in sequence near the termini of an oligomer are generallypreferable to variations in the interior, and if present are typicallywithin about 6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′terminus.

The terms “cell penetrating peptide” or “CPP” are used interchangeablyand refer to cationic cell penetrating peptides, also called transportpeptides, carrier peptides, or peptide transduction domains. Thepeptides, as shown herein, have the capability of inducing cellpenetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cellsof a given cell culture population, including all integers in between,and allow macromolecular translocation within multiple tissues in vivoupon systemic administration.

The terms “antisense oligomer” or “antisense compound” or “antisenseoligonucleotide” or “oligonucleotide” are used interchangeably and referto a sequence of cyclic subunits, each bearing a base-pairing moiety,linked by intersubunit linkages that allow the base-pairing moieties tohybridize to a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence. The cyclic subunits may be based on riboseor another pentose sugar or, in certain embodiments, a morpholino group(see description of morpholino oligomers below). Also contemplated arepeptide nucleic acids (PNAs), locked nucleic acids (LNAs), and2′-O-Methyl oligonucleotides, and other antisense agents known in theart.

Such an antisense oligomer can be designed to block or inhibittranslation of mRNA or to inhibit natural pre-mRNA splice processing, orinduce degradation of targeted mRNAs, and may be said to be “directedto” or “targeted against” a target sequence with which it hybridizes. Incertain embodiments, the target sequence is a region surrounding orincluding an AUG start codon of an mRNA, a 3′ or 5′ splice site of apre-processed mRNA, or a branch point. The target sequence may be withinan exon or within an intron or a combination. The target sequence for asplice site may include an mRNA sequence having its 5′ end 1 to about 25base pairs downstream of a normal splice acceptor junction in apreprocessed mRNA. A preferred target sequence for a splice is anyregion of a preprocessed mRNA that includes a splice site or iscontained entirely within an exon coding sequence or spans a spliceacceptor or donor site. An oligomer is more generally said to be“targeted against” a biologically relevant target such as, in thepresent invention, a human LMNA gene pre-mRNA encoding the lamin Aprotein, when it is targeted against the nucleic acid of the target inthe manner described above. Exemplary targeting sequences include SEQ IDNOS: 3-34

Included are antisense oligonucleotides that comprise, consistessentially of, or consist of one or more of SEQ ID NOS:3-34. Alsoincluded are variants of these antisense oligomers, including variantoligomers having 80%, 85%, 90%, 95%, 97%, 98%, or 99% (including allintegers in between) sequence identity or sequence homology to any oneof SEQ ID NOS:3-34, and/or variants that differ from these sequences byabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, preferably thosevariants that modulate progerin expression in a cell. Also included areoligonucleotides of any one or more of SEQ ID NOS: 3-34, which comprisea suitable number of cationic or other modified linkages, as describedherein, e.g., up to about 1 per every 2-5 uncharged linkages, such asabout 4-5 per every 10 uncharged linkages, and/or which comprise anArg-rich cell-penetrating transport peptide attached thereto, as alsodescribed herein.

The terms “morpholino oligomer” or “PMO” (phosphoramidate- orphosphorodiamidate morpholino oligomer) refer to an oligonucleotideanalog composed of morpholino subunit structures, where (i) thestructures are linked together by phosphorus-containing linkages, one tothree atoms long, preferably two atoms long, and preferably uncharged orcationic, joining the morpholino nitrogen of one subunit to a 5′exocyclic carbon of an adjacent subunit, and (ii) each morpholino ringbears a purine or pyrimidine or an equivalent base-pairing moietyeffective to bind, by base specific hydrogen bonding, to a base in apolynucleotide. Variations can be made to this linkage as long as theydo not interfere with binding or activity. For example, the oxygenattached to phosphorus may be substituted with sulfur(thiophosphorodiamidate). The 5′ oxygen may be substituted with amino orlower alkyl substituted amino. The pendant nitrogen attached tophosphorus may be unsubstituted, monosubstituted, or disubstituted with(optionally substituted) lower alkyl. See also the discussion ofcationic linkages below. The purine or pyrimidine base pairing moiety istypically adenine, cytosine, guanine, uracil, thymine or inosine. Thesynthesis, structures, and binding characteristics of morpholinooligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, and PCT Appn.Nos. PCT/US07/11435 (cationic linkages) and U.S. Ser. No. 08/012,804(improved synthesis), all of which are incorporated herein by reference.

“PMO+” refers to phosphorodiamidate morpholino oligomers comprising anynumber of (1-piperazino)phosphinylideneoxy,(1-(4-(ω-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages (A2and A3) that have been described previously (see e.g., PCT publicationWO/2008/036127 which is incorporated herein by reference in itsentirety.

“PMO-X” refers to phosphorodiamidate morpholino oligomers disclosedherein comprising at least one (B) linkage or at least one of thedisclosed terminal modifications, and as disclosed in WO2011/150408 andUS2012/0065169, which are incorporated herein by reference in theirentireties. Further PMO-X phosphorodiamidate morpholino oligomers usefulherein may be found in U.S. Provisional Application No. 61/561,806,filed Nov. 18, 2011, which is incorporated herein by reference in itsentirety.

A “phosphoramidate” group comprises phosphorus having three attachedoxygen atoms and one attached nitrogen atom, while a“phosphorodiamidate” group comprises phosphorus having two attachedoxygen atoms and two attached nitrogen atoms. In the uncharged or themodified intersubunit linkages of the oligomers described herein andco-pending U.S. Patent Application No. 61/349,783 and Ser. No.11/801,885, one nitrogen is always pendant to the backbone chain. Thesecond nitrogen, in a phosphorodiamidate linkage, is typically the ringnitrogen in a morpholino ring structure.

“Thiophosphoramidate” or “thiophosphorodiamidate” linkages arephosphoramidate or phosphorodiamidate linkages, respectively, whereinone oxygen atom, typically the oxygen pendant to the backbone, isreplaced with sulfur.

“Intersubunit linkage” refers to the linkage connecting two morpholinosubunits, for example structure (I).

“Charged”, “uncharged”, “cationic” and “anionic” as used herein refer tothe predominant state of a chemical moiety at near-neutral pH, e.g.,about 6 to 8. For example, the term may refer to the predominant stateof the chemical moiety at physiological pH, that is, about 7.4.

The term “oligonucleotide analog” refers to an oligonucleotide having(i) a modified backbone structure, e.g., a backbone other than thestandard phosphodiester linkage found in natural oligo- andpolynucleotides, and (ii) optionally, modified sugar moieties, e.g.,morpholino moieties rather than ribose or deoxyribose moieties.Oligonucleotide analogs support bases capable of hydrogen bonding byWatson-Crick base pairing to standard polynucleotide bases, where theanalog backbone presents the bases in a manner to permit such hydrogenbonding in a sequence-specific fashion between the oligonucleotideanalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). Preferred analogs are thosehaving a substantially uncharged, phosphorus containing backbone.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 50-100%, typically at least 60% to 100% or 75%or 80% of its linkages, are uncharged, and contain a single phosphorousatom. Antisense oligonucleotides and oligonucleotide analogs may containbetween about 8 and 40 subunits, typically about 8-25 subunits, andpreferably about 12 to 25 subunits (including all integers and ranges inbetween). In certain embodiments, oligonucleotides may have exactsequence complementarity to the target sequence or near complementarity,as defined below.

A “subunit” of an oligonucleotide refers to one nucleotide (ornucleotide analog) unit. The term may refer to the nucleotide unit withor without the attached intersubunit linkage, although, when referringto a “charged subunit”, the charge typically resides within theintersubunit linkage (e.g., a phosphate or phosphorothioate linkage or acationic linkage).

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g., 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses of the oligomers, Tsand Us are interchangeable. For instance, with other antisensechemistries such as 2′-O-methyl antisense oligonucleotides that are moreRNA-like, the T bases may be shown as U (see, e.g., sequence listing).

An “amino acid subunit” or “amino acid residue” can refer to an α-aminoacid residue (—CO—CHR—NH—) or a β- or other amino acid residue (e.g.,—CO—(CH₂)_(n)CHR—NH—), where R is a side chain (which may includehydrogen) and n is 1 to 7, preferably 1 to 4.

The term “naturally occurring amino acid” refers to an amino acidpresent in proteins found in nature, such as the 20 (L)-amino acidsutilized during protein biosynthesis as well as others such as4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine,citrulline and ornithine. The term “non-natural amino acids” refers tothose amino acids not present in proteins found in nature, examplesinclude beta-alanine (β-Ala), 6-aminohexanoic acid (Ahx) and6-aminopentanoic acid. Additional examples of “non-natural amino acids”include, without limitation, (D)-amino acids, norleucine, norvaline,p-fluorophenylalanine, ethionine and the like, which are known to aperson skilled in the art.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide” or “isolated oligonucleotide,” asused herein, may refer to a polynucleotide that has been purified orremoved from the sequences that flank it in a naturally-occurring state,e.g., a DNA fragment that has been removed from the sequences that arenormally adjacent to the fragment.

An “effective amount” or “therapeutically effective amount” refers to anamount of therapeutic compound, such as an antisense oligomer,administered to a mammalian subject, either as a single dose or as partof a series of doses, which is effective to produce a desiredtherapeutic effect (e.g., sensitization of a cancer cell to achemotherapeutic) For an antisense oligomer, this effect is typicallybrought about by inhibiting translation or natural splice-processing ofa selected target sequence. An “effective amount,” targeted against LMNAmRNA, also relates to an amount effective to modulate expression ofprogerin.

By “enhance” or “enhancing,” or “increase” or “increasing,” or“stimulate” or “stimulating,” refers generally to the ability of one orantisense compounds or compositions to produce or cause a greaterphysiological response (i.e., downstream effects) in a cell, as comparedto the response caused by either no antisense compound or a controlcompound. An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1), e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amountproduced by no antisense compound (the absence of an agent) or a controlcompound.

The term “reduce” or “inhibit” may relate generally to the ability ofone or more antisense compounds of the invention to “decrease” arelevant physiological or cellular response, as measured according toroutine techniques in the diagnostic art. Relevant physiological orcellular responses (in vivo or in vitro) will be apparent to personsskilled in the art, and may include, for example, reductions inexpression of progerin as measured by mRNA and/or protein levels. A“decrease” in a response may be “statistically significant” as comparedto the response produced by no antisense compound or a controlcomposition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease,including all integers in between.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide or antisense agent is directed, that is, thesequence to which the oligonucleotide will hybridize by Watson-Crickbase pairing of a complementary sequence. In certain embodiments, thetarget sequence may be a contiguous region of a pre-mRNA that includesboth intron and exon target sequence. In certain other embodiments, thetarget sequence will consist exclusively of either intron or exonsequences.

The term “targeting sequence” or “antisense targeting sequence” refersto the sequence in an oligonucleotide or other antisense agent that iscomplementary (meaning, in addition, substantially complementary) to thetarget sequence in the RNA genome. The entire sequence, or only aportion, of the antisense compound may be complementary to the targetsequence. For example, in an oligonucleotide having 20-30 bases, about6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, or 29 may be targeting sequences that are complementaryto the target region. Typically, the targeting sequence is formed ofcontiguous bases, but may alternatively be formed of non-contiguoussequences that when placed together, e.g., from opposite ends of theoligonucleotide, constitute sequence that spans the target sequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be functionally “complementary.” Incertain embodiments, an oligonucleotide may have at most one mismatchwith the target sequence out of 10 nucleotides, and preferably at mostone mismatch out of 20. Alternatively, an oligonucleotide may have atleast 90% sequence homology, and preferably at least 95% sequencehomology, with the exemplary antisense targeting sequences describedherein.

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to the target under physiological conditions,with a Tm substantially greater than 45° C., preferably at least 50° C.,and typically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Again, suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395). In this way sequences ofa similar or substantially different length to those cited herein couldbe compared by insertion of gaps into the alignment, such gaps beingdetermined, for example, by the comparison algorithm used by GAP.

The terms “sequence identity” or, for example, comprising a “sequence50% identical to,” as used herein, refer to the extent that sequencesare identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 8 or 10 but frequently 15 to 18 and often at least 25 monomerunits, inclusive of nucleotides and amino acid residues, in length.Because two polynucleotides may each comprise (1) a sequence (i.e., onlya portion of the complete polynucleotide sequence) that is similarbetween the two polynucleotides, and (2) a sequence that is divergentbetween the two polynucleotides, sequence comparisons between two (ormore) polynucleotides are typically performed by comparing sequences ofthe two polynucleotides over a “comparison window” to identify andcompare local regions of sequence similarity. A “comparison window”refers to a conceptual segment of at least 6 contiguous positions,usually about 50 to about 100, more usually about 100 to about 150 inwhich a sequence is compared to a reference sequence of the same numberof contiguous positions after the two sequences are optimally aligned.The comparison window may comprise additions or deletions (i.e., gaps)of about 20% or less as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences.

Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

An agent is “actively taken up by mammalian cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport,” referring to transport of agents across a mammalian cellmembrane by e.g., an ATP-dependent transport mechanism, or by“facilitated transport,” referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane. For both active and facilitatedtransport, oligonucleotide analogs preferably have a substantiallyuncharged backbone, as defined below.

A “heteroduplex” refers to a duplex between an antisense oligonucleotideand the complementary portion of a target RNA. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, such as RNaseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

As used herein, the term “body fluid” encompasses a variety of sampletypes obtained from a subject including, urine, saliva, plasma, blood,spinal fluid, or other sample of biological origin, such as skin cellsor dermal debris, and may refer to cells or cell fragments suspendedtherein, or the liquid medium and its solutes.

The term “relative amount” is used where a comparison is made between atest measurement and a control measurement. The relative amount of areagent forming a complex in a reaction is the amount reacting with atest specimen, compared with the amount reacting with a controlspecimen. The control specimen may be run separately in the same assay,or it may be part of the same sample (for example, normal tissuesurrounding a malignant area in a tissue section).

“Treatment” of an individual or a cell is any type of interventionprovided as a means to alter the natural course of a disease orpathology in the individual or cell. Treatment includes, but is notlimited to, administration of, e.g., a pharmaceutical composition, andmay be performed either prophylactically, or subsequent to theinitiation of a pathologic event or contact with an etiologic agent.Treatment includes any desirable effect on the symptoms or pathology ofa disease or condition associated with inflammation, among othersdescribed herein.

Also included are “prophylactic” treatments, which can be directed toreducing the rate of progression of the disease or condition beingtreated, delaying the onset of that disease or condition, or reducingthe severity of its onset. “Treatment” or “prophylaxis” does notnecessarily indicate complete eradication, cure, or prevention of thedisease or condition, or associated symptoms thereof.

A wild-type gene or gene product is that which is most frequentlyobserved in a population and is thus arbitrarily designed the “normal”or “wild-type” form of the gene.

The chemical terms below have the following meanings, unless indicatedotherwise:

“Amino” refers to the —NH₂ radical.

“Cyano” or “nitrile” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Guanidinyl” refers to the —NHC(═NH)NH₂ substituent.

“Amidinyl” refers to the —C(═NH)NH₂ substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Cholate” refers to the following structure:

“Deoxycholate” refers to the following structure:

“Alkyl” refers to a straight or branched hydrocarbon chain radical whichis saturated or unsaturated (i.e., contains one or more double and/ortriple bonds), having from one to thirty carbon atoms, and which isattached to the rest of the molecule by a single bond. Alkyls comprisingany number of carbon atoms from 1 to 30 are included. An alkylcomprising up to 30 carbon atoms is referred to as a C₁-C₃₀ alkyl,likewise, for example, an alkyl comprising up to 12 carbon atoms is aC₁-C₁₂ alkyl. Alkyls (and other moieties defined herein) comprisingother numbers of carbon atoms are represented similarly. Alkyl groupsinclude, but are not limited to, C₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₁-C₁₅alkyl, C₁-C₁₀ alkyl, C₁-C₈ alkyl, C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl,C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈ alkyl and C₄-C₈ alkyl. Representativealkyl groups include, but are not limited to, methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, i-butyl, s-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl,propynyl, but-2-ynyl, but-3-ynyl, pentynyl, hexynyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup may be optionally substituted as described below.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group.Alkylenes may be saturated or unsaturated (i.e., contains one or moredouble and/or triple bonds). Representative alkylenes include, but arenot limited to, C₁-C₁₂ alkylene, C₁-C₈ alkylene, C₁-C₆ alkylene, C₁-C₄alkylene, C₁-C₃ alkylene, C₁-C₂ alkylene, C₁ alkylene. Representativealkylene groups include, but are not limited to, methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted as described below.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined. Unless stated otherwise specifically in thespecification, an alkoxy group may be optionally substituted asdescribed below.

Alkoxyalkyl” refers to a radical of the formula —R_(b)OR_(a) where R_(a)is an alkyl radical as defined and where R_(b) is an alkylene radical asdefined. Unless stated otherwise specifically in the specification, analkoxyalkyl group may be optionally substituted as described below.

“Alkylcarbonyl” refers to a radical of the formula —C(═O)R_(a) whereR_(a) is an alkyl radical as defined above. Unless stated otherwisespecifically in the specification, an alkylcarbonyl group may beoptionally substituted as described below.

“Alkyloxycarbonyl” refers to a radical of the formula —C(═O)OR_(a) whereR_(a) is an alkyl radical as defined. Unless stated otherwisespecifically in the specification, an alkyloxycarbonyl group may beoptionally substituted as described below.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined above.Unless stated otherwise specifically in the specification, an alkylaminogroup may be optionally substituted as described below.

“Amidyl” refers to a radical of the formula —N(H)C(═O) R_(a) where R_(a)is an alkyl or aryl radical as defined herein. Unless stated otherwisespecifically in the specification, an amidyl group may be optionallysubstituted as described below.

“Amidinylalkyl” refers a radical of the formula —R_(b)—C(═NH)NH₂ whereR_(b) is an alkylene radical as defined above. Unless stated otherwisespecifically in the specification, an amidinylalkyl group may beoptionally substituted as described below.

“Amidinylalkylcarbonyl” refers a radical of the formula—C(═O)R_(b)—C(═NH)NH₂ where R_(b) is an alkylene radical as definedabove. Unless stated otherwise specifically in the specification, anamidinylalkylcarbonyl group may be optionally substituted as describedbelow.

“Aminoalkyl” refers to a radical of the formula —R_(b)—NR_(a)R_(a) whereR_(b) is an alkylene radical as defined above, and each R_(a) isindependently a hydrogen or an alkyl radical.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above. Unless stated otherwise specifically inthe specification, a thioalkyl group may be optionally substituted.

“Aryl” refers to a radical derived from a hydrocarbon ring systemcomprising hydrogen, 6 to 30 carbon atoms and at least one aromaticring. The aryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl radicals include, but are not limited to, aryl radicalsderived from the hydrocarbon ring systems of aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane,indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, andtriphenylene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals that are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl, trityland the like. Unless stated otherwise specifically in the specification,an aralkyl group may be optionally substituted.

“Arylcarbonyl” refers to a radical of the formula —C(═O)R_(c) whereR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anarylcarbonyl group may be optionally substituted.

“Aryloxycarbonyl” refers to a radical of the formula —C(═O)OR_(c) whereR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anaryloxycarbonyl group may be optionally substituted.

“Aralkylcarbonyl” refers to a radical of the formula —C(═O)R_(b)—R_(c)where R_(b) is an alkylene chain as defined above and R_(c) is one ormore aryl radicals as defined above, for example, phenyl. Unless statedotherwise specifically in the specification, an aralkylcarbonyl groupmay be optionally substituted.

“Aralkyloxycarbonyl” refers to a radical of the formula—C(═O)OR_(b)—R_(c) where R_(b) is an alkylene chain as defined above andR_(c) is one or more aryl radicals as defined above, for example,phenyl. Unless stated otherwise specifically in the specification, anaralkyloxycarbonyl group may be optionally substituted.

“Aryloxy” refers to a radical of the formula —OR_(c) where R_(c) is oneor more aryl radicals as defined above, for example, phenyl. Unlessstated otherwise specifically in the specification, an arylcarbonylgroup may be optionally substituted.

“Cycloalkyl” refers to a stable, non-aromatic, monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems, whichis saturated or unsaturated, and attached to the rest of the molecule bya single bond. Representative cycloalkyls include, but are not limitedto, cycloaklyls having from three to fifteen carbon atoms and from threeto eight carbon atoms, Monocyclic cyclcoalkyl radicals include, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Polycyclic radicals include, for example, adamantyl,norbornyl, decalinyl, and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unlessotherwise stated specifically in the specification, a cycloalkyl groupmay be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Cycloalkylcarbonyl” refers to a radical of the formula —C(═O)R_(d)where R_(d) is a cycloalkyl radical as defined above. Unless statedotherwise specifically in the specification, a cycloalkylcarbonyl groupmay be optionally substituted.

Cycloalkyloxycarbonyl” refers to a radical of the formula —C(═O)OR_(d)where R_(d) is a cycloalkyl radical as defined above. Unless statedotherwise specifically in the specification, a cycloalkyloxycarbonylgroup may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure. When the fused ring is a heterocyclyl ringor a heteroaryl ring, any carbon atom on the existing ring structurewhich becomes part of the fused heterocyclyl ring or the fusedheteroaryl ring may be replaced with a nitrogen atom.

“Guanidinylalkyl” refers a radical of the formula —R_(b)—NHC(═NH)NH₂where R_(b) is an alkylene radical as defined above. Unless statedotherwise specifically in the specification, a guanidinylalkyl group maybe optionally substituted as described below.

“Guanidinylalkylcarbonyl” refers a radical of the formula—C(═O)R_(b)—NHC(═NH)NH₂ where R_(b) is an alkylene radical as definedabove. Unless stated otherwise specifically in the specification, aguanidinylalkylcarbonyl group may be optionally substituted as describedbelow.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl,2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl,1,2-dibromoethyl, and the like. Unless stated otherwise specifically inthe specification, a haloalkyl group may be optionally substituted.

“Perhalo” or “perfluoro” refers to a moiety in which each hydrogen atomhas been replaced by a halo atom or fluorine atom, respectively.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to24-membered non-aromatic ring radical comprising 2 to 23 carbon atomsand from one to 8 heteroatoms selected from the group consisting ofnitrogen, oxygen, phosphorous and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl,1,1-dioxo-thiomorpholinyl, 12-crown-4, 15-crown-5, 18-crown-6,21-crown-7, aza-18-crown-6, diaza-18-crown-6, aza-21-crown-7, anddiaza-21-crown-7. Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen,phosphorous and sulfur, and at least one aromatic ring. For purposes ofthis invention, the heteroaryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

All the above groups may be either substituted or unsubstituted. Theterm “substituted” as used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl,alkylamino, amidyl, amidinylalkyl, amidinylalkylcarbonyl, aminoalkyl,aryl, aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl,aralkyloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl,cycloalkylcarbonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl,guanidinylalkyl, guanidinylalkylcarbonyl, haloalkyl, heterocyclyl and/orheteroaryl), may be further functionalized wherein at least one hydrogenatom is replaced by a bond to a non-hydrogen atom substituent. Unlessstated specifically in the specification, a substituted group mayinclude one or more substituents selected from: oxo, —CO₂H, nitrile,nitro, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl,alkylcarbonyl, alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl,aryloxycarbonyl, aralkylcarbonyl, aralkyloxycarbonyl, aryloxy,cycloalkyl, cycloalkylalkyl, cycloalkylcarbonyl,cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl,heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such astrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example,trifluoromethyl or trifluoromethoxy. “Substituted” also means any of theabove groups in which one or more hydrogen atoms are replaced by ahigher-order bond (e.g., a double- or triple-bond) to a heteroatom suchas oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen ingroups such as imines, oximes, hydrazones, and nitriles. For example,“substituted” includes any of the above groups in which one or morehydrogen atoms are replaced with —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h), —SH, —SR_(g) or—SSR_(g). In the foregoing, R_(g) and R_(h) are the same or differentand independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. In addition, each of the foregoing substituents mayalso be optionally substituted with one or more of the abovesubstituents. Furthermore, any of the above groups may be substituted toinclude one or more internal oxygen or sulfur atoms. For example, analkyl group may be substituted with one or more internal oxygen atoms toform an ether or polyether group. Similarly, an alkyl group may besubstituted with one or more internal sulfur atoms to form a thioether,disulfide, etc. Amidyl moieties may be substituted with up to 2 haloatoms, while other groups above may be substituted with one or more haloatoms. With the exception of alkyl groups, all other groups may also besubstituted with amino or monoalklyamino. With the exception of alkyland alkylcarbonyl groups, all other groups may also be substituted withguanidinyl or amidynyl. Optional substituents for any of the abovegroups also include arylphosphoryl, for example —R_(a)P(Ar)₃ wherein Rais an alkylene and Ar is aryl moiety, for example phenyl.

“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, asexemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl,n-pentyl, and isopentyl. In certain embodiments, a “lower alkyl” grouphas one to four carbon atoms. In other embodiments a “lower alkyl” grouphas one to two carbon atoms; i.e. methyl or ethyl. Analogously, “loweralkenyl” refers to an alkenyl radical of two to six, preferably three orfour, carbon atoms, as exemplified by allyl and butenyl.

A “non-interfering” substituent is one that does not adversely affectthe ability of an antisense oligomer as described herein to bind to itsintended target. Such substituents include small and/or relativelynon-polar groups such as methyl, ethyl, methoxy, ethoxy, or fluoro.

LMNA Targeting

Examples for use in the methods of the invention include antisenseoligonucleotides that target SEQ ID NOs: 1 and/or 2, discussed below.

Certain antisense oligonucleotides for use in the methods of theinvention may comprise a targeting sequence that is complementary to oneor more bases of exon 11 in the human LMNA gene including the wild-typesequence (SEQ ID NO: 1) and/or the sequence found in HGPS patients, asshown in SEQ ID NO: 2. These target sequences are shown in Table 1below:

TABLE 1 Exemplary LMNA Target Sequences NAME SEQUENCE SEQ ID NO:LMNA exon 11 GGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTG 1CGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGC GGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAG HGPS exon 11GGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTG 2CGCTCGCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGT GGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCAACTCCAGCCCCCGAACCCAG

Examples include antisense oligonucleotides that are fully complementaryto LMNA exon 11 (SEQ ID NO: 1 or 2) including those that are alsocomplementary to the cryptic splice site of LMNA exon 11 underlined inSEQ ID NO: 1 and 2 in Table 1

(e.g., CAGGTGGGC/T).Certain antisense oligonucleotides may comprise a targeting sequencewhere the 3′-most base is complementary to a base in LMNA exon 11 thatis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bases downstream of theunderlined cryptic splice site in SEQ ID NO: 1 or 2 (see Table 1), orwhich is complementary to a base in LMNA exon 11 that is 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39 or 40 upstream of the cryptic splicesite. Exemplary oligonucleotides of this type are among those listedbelow as SEQ ID NOs: 3-16. Certain antisense oligonucleotides maycomprise a targeting sequence where the 3′-most base is complementary toa base in LMNA intron 10 that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 bases upstream of LMNAexon 11 SEQ ID NO: 1 or 2 (see Table 1). Certain antisenseoligonucleotides may comprise a targeting sequence where the 3′-mostbase is complementary to a base in LMNA exon 10 that is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 bases upstream of LMNA intron 10. Certainembodiments relate to the use of a combination of these antisenseagents. Specific embodiments include antisense oligonucleotidescomprising all or a portion (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 bases) of SEQ ID NO: 1 and/or 2.

Antisense oligonucleotides for use in the methods of the invention canalso be targeted against, or be complementary to, a variety of region(s)in a pre-processed LMNA mRNA, such as an exon, an intron, an exon-intronjunction, or a splice junction. For instance, certain antisenseoligonucleotides may comprise a targeting sequence that is complementaryto a region (target sequence) that overlaps the splice junction of asplice donor (SD) or splice acceptor (SA) sites of exons 10 and 11 ofLMNA pre-mRNA, and is complementary to a portion of an exonic region(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleotides) and a portion of an intronicregion (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24 or 25 nucleotides) of the pre-processed mRNA. Exemplary targetingsequences of the above types are listed below as SEQ ID NOs 17-34.

Selected antisense targeting sequences can be made shorter, e.g., about12 bases, or longer, e.g., about 40 bases, and include a small number ofmismatches, as long as the sequence is sufficiently complementary toeffect splicing, and/or other form of inhibition upon hybridization withthe target, and forms with the target RNA, a heteroduplex having a Tm of45° C. or greater.

In certain embodiments, the degree of complementarity between the targetand antisense targeting sequence is sufficient to form a stable duplex.The region of complementarity of the antisense oligomers with the targetRNA sequence may be as short as 8-11 bases, but is preferably 12-15bases or more, e.g., 12-20 bases, 12-25, or 15-25 bases, including allintegers and ranges in between these ranges. An antisense oligomer ofabout 14-15 bases is generally long enough to have a uniquecomplementary sequence in the target mRNA. In certain embodiments, aminimum length of complementary bases may be required to achieve therequisite binding Tm, as discussed below.

In certain embodiments, oligomers as long as 40 bases may be suitable,where at least a minimum number of bases, e.g., 10-12 bases, arecomplementary to the target sequence. In general, however, facilitatedor active uptake in cells is optimized at oligomer lengths less thanabout 30. For PMO oligomers, described further below, an optimum balanceof binding stability and uptake generally occurs at lengths of 18-30bases. Included are antisense oligomers (e.g., PNAs, LNAs, 2′-OMe, MOE,PMOs) that consist of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, or 40 bases, in which at least about 6, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 contiguous and/or non-contiguous basesare complementary to a target sequence described herein, including thetarget sequences of SEQ ID NOs: 1 and/or 2, or variants thereof.

In certain embodiments, antisense oligomers for use in the methods ofthe invention may be 100% complementary to the LMNA pre-mRNA nucleicacid target sequence, or they may include mismatches, e.g., toaccommodate variants, as long as a heteroduplex formed between theoligomer and the target sequence is sufficiently stable to withstand theaction of cellular nucleases and other modes of degradation ordisplacement which may occur in vivo. Oligomer backbones which are lesssusceptible to cleavage by nucleases are discussed below. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pairs in theduplex, and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability. Although such anantisense oligomer is not necessarily 100% complementary to the targetsequence, it is effective to stably and specifically bind to the targetsequence, such that a biological activity of the nucleic acid target,e.g., expression of the progerin protein(s), is modulated.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an antisense compoundwith respect to complementary-sequence RNA may be measured byconventional methods, such as those described by Hames et al., NucleicAcid Hybridization, IRL Press, 1985, pp. 107-108 or as described inMiyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridizationtechniques, Methods Enzymol. Vol. 154 pp. 94-107. In certainembodiments, antisense oligomer may have a binding Tm, with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. Tm's in the range 60-80° C. or greaterare preferred. According to well known principles, the Tm of an oligomercompound, with respect to a complementary-based RNA hybrid, can beincreased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high Tm (50° C. or greater) at a length of 25 basesor less are generally preferred over those requiring greater than 25bases for high Tm values.

In certain embodiments, such as PMO oligomers, the antisense activity ofan oligomer may be enhanced by using a mixture of uncharged and cationicphosphorodiamidate linkages, as exemplified in FIG. 1C. The total numberof cationic linkages in the oligomer can vary from 1 to 10 (includingall integers in between), and be interspersed throughout the oligomer.Preferably the number of charged linkages is at least 2 and no more thanhalf the total backbone linkages, e.g., between 2, 3, 4, 5, 6, 7, or 8positively charged linkages, and preferably each charged linkage isseparated along the backbone by at least 1, 2, 3, 4, or 5 unchargedlinkages. A preferred cationic linkage of the invention includes the apnlinkage B10 as shown in Table 3.

Exemplary antisense sequences for use in the methods of the inventionfor targeting the human LMNA pre-mRNA are shown in Table 1 below.Antisense oligonucleotides can comprise all or a portion of thesetargeting sequences.

TABLE 2 Exemplary HGPS Targeting Sequences* SEQ ID PMO nameTargeting Sequence 5′-3′ NO: Exo11.25.133 CCGCTGGCAGATGCCTTGTCGGCAG  3Exo11.25.138 CTGAGCCGCTGGCAGATGCCTTGTC  4 Exo11.25.142GCTCCTGAGCCGCTGGCAGATGCCT  5 Exo11.25.145 TGGGCTCCTGAGCCGCTGGCAGATG  6Exo11.25.149 CACCTGGGCTCCTGAGCCGCTGGCA  7 Exo11.25.154CCACCCACCTGGGCTCCTGAGCCGC  8 Exo11.25.158 GGGTCCACCCACCTGGGCTCCTGAG  9Exo11.25.162 AGATGGGTCCACCCACCTGGGCTCC 10 Exo11.25.166GAGGAGATGGGTCCACCCACCTGGG 11 Exo11.25.170 GCCAGAGGAGATGGGTCCACCCACC 12Exo11.25.174 AAGAGCCAGAGGAGATGGGTCCACC 13 Exo11.25.177CAGAAGAGCCAGAGGAGATGGGTCC 14 Exo11.25.181 GAGGCAGAAGAGCCAGAGGAGATGG 15Exo11.25.185 ACTGGAGGCAGAAGAGCCAGAGGAG 16 Exo10SD.25.69ACGTGGTGGTGATGGAGCAGGTCAT 17 Exo10SD.25.73 ACTCACGTGGTGGTGATGGAGCAGG 18Exo10SD.25.79 GCTACCACTCACGTGGTGGTGATGG 19 Exo10SD.25.84CGGCGGCTACCACTCACGTGGTGGT 20 Exo10SD.25.87 CAGCGGCGGCTACCACTCACGTGGT 21Exo10SD.25.90 CCTCAGCGGCGGCTACCACTCACGT 22 Exo10SD.25.92GGCCTCAGCGGCGGCTACCACTCAC 23 Exo10SD.25.96 GCTCGGCCTCAGCGGCGGCTACCAC 24Exo11SA.25.779 CGAGTCTGGGACTGACCACTCAGGC 25 Exo11SA.25.796AGGCTCAGGCGGGACGGCGAGTCTG 26 Exo11SA.25.801 AGACAAGGCTCAGGCGGGACGGCGA 27Exo11SA.25.805 AGGGAGACAAGGCTCAGGCGGGACG 28 Exo11SA.25.809GGGAAGGGAGACAAGGCTCAGGCGG 29 Exo11SA.25.814 GCCCTGGGAAGGGAGACAAGGCTCA 30Exo11SA.25.820 GTGGGAGCCCTGGGAAGGGAGACAA 31 ExollSA.25.828CTGCTGCAGTGGGAGCCCTGGGAAG 32 Exo11SA.25.830 AGCTGCTGCAGTGGGAGCCCTGGGA 33Exo11SA.25.836 CCCCCGAGCTGCTGCAGTGGGAGCC 34 HsEx10GCTACCACTCACGTGGTGGTGATGG-AcR₆G 35 HsEx11GGGTCCACCCACCTGGGCTCCTGAG-AcR₆G 36 HsEx10-apn GC^(apn) TACCAC^(apn)TCACG^(apn) TGGTGG^(apn) TGATGG 37 HsEx11-apn GGG^(apn) TCCACCCACC^(apn)TGGGC^(apn) TCC^(apn) TGAG 38 *AcR₆G denotes a preferredcell-penetrating peptide transporter (acylated R₆; SEQ ID NO: 45)conjugated with a glycine linker to the 3′ end of an exemplary targetingsequence. The ^(apn) T in SEQ ID NOs 19 and 20 refer to an apnintersubunit linkage as further described below in Table 3, linkage B10.

Antisense Oligonucleotide Compounds for Use in Methods of Invention

The antisense oligonucleotides for use in the methods of the presentinvention typically (a) have the ability to be actively taken up bymammalian cells, and (b) once taken up, form a duplex with the targetRNA with a Tm greater than about 45° C. In certain embodiments, theoligomer backbone may be substantially uncharged, and, preferably, maybe recognized as a substrate for active or facilitated transport acrossthe cell membrane. The ability of the oligomer to form a stable duplexwith the target RNA may also relate to other features of the oligomerbackbone, including the length and degree of complementarity of theantisense oligomer with respect to the target, the ratio of G:C to A:Tbase matches, and the positions of any mismatched bases. The ability ofthe antisense oligomer to resist cellular nucleases may promote survivaland ultimate delivery of the agent to the cell cytoplasm. Included areantisense oligomers composed of PMO, PMO+ (PMOplus), PMO-X, LNA, PNAs,and/or 2′O-Me-based chemistries, described herein. In general, PNA andLNA chemistries utilize shorter targeting oligomers due to theirrelatively high target binding strength compared to PMO and 2′O-Meoligomers.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm, Buchardtet al. 1993). The backbone of PNAs is formed by peptide bonds ratherthan phosphodiester bonds, making them well-suited for antisenseapplications (see structure below). The backbone is uncharged, resultingin PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermalstability. PNAs are not recognized by nucleases or proteases.

PNAs are produced synthetically using any technique known in the art.PNA is a DNA analog in which a polyamide backbone replaces thetraditional phosphate ribose ring of DNA as shown below.

Despite a radical structural change to the natural RNA or DNA structure,PNA is capable of sequence-specific binding in a helix form to DNA orRNA. Characteristics of PNA include a high binding affinity tocomplementary DNA or RNA, a destabilizing effect caused by single-basemismatch, resistance to nucleases and proteases, hybridization with DNAor RNA independent of salt concentration and triplex formation withhomopurine DNA. Panagene™ has developed its proprietary Bts PNA monomers(Bts; benzothiazole-2-sulfonyl group) and proprietary oligomerisationprocess. The PNA oligomerisation using Bts PNA monomers is composed ofrepetitive cycles of deprotection, coupling and capping. Panagene'spatents to this technology include U.S. Pat. No. 6,969,766, U.S. Pat.No. 7,211,668, U.S. Pat. No. 7,022,851, U.S. Pat. No. 7,125,994, U.S.Pat. No. 7,145,006 and U.S. Pat. No. 7,179,896. Representative UnitedStates patents that teach the preparation of PNA compounds include, butare not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,each of which is herein incorporated by reference. Further teaching ofPNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.

Oligonucleotide compounds for use in the methods of the invention mayalso contain “locked nucleic acid” subunits (LNAs). The structures ofLNAs are known in the art: for example, Wengel, et al., ChemicalCommunications (1998) 455; Tetrahedron (1998) 54, 3607, and Accounts ofChem. Research (1999) 32, 301); Obika, et al., Tetrahedron Letters(1997) 38, 8735; (1998) 39, 5401, and Bioorganic Medicinal Chemistry(2008)16, 9230. Exemplary, non-limiting LNA structures are illustratedbelow:

Compounds for use in the methods of the invention may incorporate one ormore LNAs; in some cases, the compounds may be entirely composed ofLNAs. Methods for the synthesis of individual LNA nucleoside subunitsand their incorporation into oligonucleotides are known in the art: U.S.Pat. Nos. 7,572,582; 7,569,575; 7,084,125; 7,060,809; 7,053,207;7,034,133; 6,794,499; and 6,670,461. Typical intersubunit linkersinclude phosphodiester and phosphorothioate moieties; alternatively,non-phosphorous containing linkers may be employed. A preferredembodiment is an LNA containing compound where each LNA subunit isseparated by a DNA subunit (i.e., a deoxyribose nucleotide). Furtherpreferred compounds are composed of alternating LNA and DNA subunitswhere the intersubunit linker is phosphorothioate.

A preferred oligomer structure for use in the methods of the inventionemploys morpholino-based subunits bearing base-pairing moieties, joinedby uncharged linkages, as described above. Especially preferred is asubstantially uncharged phosphorodiamidate-linked morpholino oligomer(PMO). Morpholino oligonucleotides, including antisense oligomers, aredetailed, for example, in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866,5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and 5,506,337,and in PCT application No. U.S. Ser. No. 08/088,339, all of which areincorporated by reference.

Certain properties of the morpholino-based subunits include: the abilityto be linked in a oligomeric form by stable, uncharged backbonelinkages; the ability to support a nucleotide base (e.g., adenine,cytosine, guanine or uracil) such that the polymer formed can hybridizewith a complementary-base target nucleic acid, including target RNA,with high Tm, even with oligomers as short as 10-14 bases; the abilityof the oligomer to be actively transported into mammalian cells; and theability of the oligomer:RNA heteroduplex to resist RNase degradation.

Properties of the morpholino-based subunits include: 1) the ability tobe linked in a oligomeric form by stable, uncharged or positivelycharged backbone linkages; 2) the ability to support a nucleotide base(e.g., adenine, cytosine, guanine, thymidine, uracil and hypoxanthine)such that the polymer formed can hybridize with a complementary-basetarget nucleic acid, including target RNA, Tm values above about 45° C.in relatively short oligonucleotides (e.g., 10-15 bases); 3) the abilityof the oligonucleotide to be actively or passively transported intomammalian cells; and 4) the ability of the antisense oligonucleotide:RNAheteroduplex to resist RNase and RNaseH degradation, respectively.

Examples of morpholino oligonucleotides for use in the methods of theinvention having phosphorus-containing backbone linkages are illustratedin FIGS. 1A-1C. A preferred phosphorodiamidate-linked morpholinooligonucleotide is shown in FIG. 1C, which is modified, in accordancewith one aspect of the present invention, to contain positively chargedgroups at preferably 10%-50% of its backbone linkages. Exemplarybackbone structures for antisense oligonucleotides of the claimedsubject matter include the morpholino subunit types shown in FIGS.1A-1C, each linked by an uncharged or positively charged,phosphorus-containing subunit linkage. FIG. 1D shows aphosphorus-containing linkage which forms the five atom repeating-unitbackbone, where the morpholino rings are linked by a 1-atom phosphoamidelinkage. FIG. 1E shows a linkage which produces a 6-atom repeating-unitbackbone. In this structure, the atom Y linking the 5′ morpholino carbonto the phosphorus group may be sulfur, nitrogen, carbon or, preferably,oxygen. The X moiety pendant from the phosphorus may be fluorine, analkyl or substituted alkyl, an alkoxy or substituted alkoxy, athioalkoxy or substituted thioalkoxy, or unsubstituted, monosubstituted,or disubstituted nitrogen, including cyclic structures, such asmorpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferablyinclude 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and arepreferably oxygen.

The linkages shown in FIGS. 1F and 1G are designed for 7-atomunit-length backbones. In FIG. 1F, the X moiety is as in FIG. 1E, andthe Y moiety may be methylene, sulfur, or, preferably, oxygen. In FIG.1G, the X and Y moieties are as FIG. 1E. Particularly preferredmorpholino oligonucleotides include those composed of morpholino subunitstructures of the form shown in FIG. 1E, where X=NH₂, N(CH₃)₂, or1-piperazine or other charged group, Y=O, and Z=O.

As noted above, the substantially uncharged oligonucleotide may bemodified, in accordance with an aspect of the invention, to includecharged linkages, e.g., up to about 1 per every 2-5 uncharged linkages,such as about 4-5 per every 10 uncharged linkages. In certainembodiments, optimal improvement in antisense activity may be seen whenabout 25% of the backbone linkages are cationic. In certain embodiments,enhancement may be seen with a small number e.g., 10-20% cationiclinkages, or where the number of cationic linkages are in the range50-80%, such as about 60%. The enhancement seen with added cationicbackbone charges may, in some cases, be further enhanced by distributingthe bulk of the charges close of the “center-region” backbone linkagesof the antisense oligonucleotide, e.g., in a 20-mer oligonucleotide with8 cationic backbone linkages, having at least 70% of these chargedlinkages localized in the 10 centermost linkages.

In certain embodiments, the antisense compounds for use in the methodsof the invention can be prepared by stepwise solid-phase synthesis,employing methods detailed in the references cited above, and below withrespect to the synthesis of oligonucleotides having a mixture oruncharged and cationic backbone linkages. In some cases, it may bedesirable to add additional chemical moieties to the antisense compound,e.g., to enhance pharmacokinetics or to facilitate capture or detectionof the compound. Such a moiety may be covalently attached, typically toa terminus of the oligomer, according to standard synthetic methods. Forexample, addition of a polyethyleneglycol moiety or other hydrophilicpolymer, e.g., one having 10-100 monomeric subunits, may be useful inenhancing solubility. One or more charged groups, e.g., anionic chargedgroups such as an organic acid, may enhance cell uptake.

A reporter moiety, such as fluorescein or a radiolabeled group, may beattached for purposes of detection. Alternatively, the reporter labelattached to the oligomer may be a ligand, such as an antigen or biotin,capable of binding a labeled antibody or streptavidin. In selecting amoiety for attachment or modification of an antisense compound, it isgenerally of course desirable to select chemical compounds of groupsthat are biocompatible and likely to be tolerated by a subject withoutundesirable side effects.

As noted above, certain of the antisense compounds for use in themethods of the invention can be constructed to contain a selected numberof cationic linkages interspersed with uncharged linkages of the typedescribed above. The intersubunit linkages, both uncharged and cationic,preferably are phosphorus-containing linkages, having the structure:

whereW is S or O, and is preferably O,X=NR¹R² or OR⁶,

Y=O or NR⁷,

and each said linkage in the oligomer is selected from:

(a) uncharged linkage (a), where each of R¹, R², R⁶ and R⁷ isindependently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), where X=NR¹R² and Y=O, and NR¹R² representsan optionally substituted piperazino group, such thatR¹R²=—CHRCHRN(R³)(R⁴)CHRCHR—, where

each R is independently H or CH₃,

R⁴ is H, CH₃, or an electron pair, and

R³ is selected from H, lower alkyl, e.g., CH₃, C(═NH)NH₂,Z-L-NHC(═NH)NH₂, and [C(O)CHR′NH]_(m)H, where: Z is C(O) or a directbond, L is an optional linker up to 18 atoms in length, preferably up to12 atoms, and more preferably up to 8 atoms in length, having bondsselected from alkyl, alkoxy, and alkylamino, R′ is a side chain of anaturally occurring amino acid or a one- or two-carbon homolog thereof,and m is 1 to 6, preferably 1 to 4;

(b2) cationic linkage (b2), where X=NR¹R² and Y=O, R¹=H or CH₃, andR²=LNR³R⁴R⁵, where L, R³, and R⁴ are as defined above, and R⁵ is H,lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), where Y=NR⁷ and X=OR⁶, and R⁷=LNR³R⁴R⁵,where L, R³, R⁴ and R⁵ are as defined above, and R⁶ is H or lower alkyl;

and at least one said linkage is selected from cationic linkages (b1),(b2), and (b3).

In certain embodiments, an oligomer may include at least two consecutivelinkages of type (a) (i.e. uncharged linkages). In further embodiments,at least 5% of the linkages in the oligomer are cationic linkages (i.e.type (b1), (b2), or (b3)); for example, 10% to 60%, and preferably20-50% linkages may be cationic linkages.

In one embodiment, at least one linkage is of type (b1), where,preferably, each R is H, R⁴ is H, CH₃, or an electron pair, and R³ isselected from H, lower alkyl, e.g., CH₃, C(═NH)NH₂, andC(O)-L-NHC(═NH)NH₂. The latter two embodiments of R³ provide a guanidinomoiety, either attached directly to the piperazine ring, or pendant to alinker group L, respectively. For ease of synthesis, the variable Z inR³ is preferably C(O) (carbonyl), as shown.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g., —CH₂—CH₂—), alkoxy (—C—O—), and alkylamino(e.g., —CH₂—NH—), with the proviso that the terminal atoms in L (e.g.,those adjacent to carbonyl or nitrogen) are carbon atoms. Althoughbranched linkages (e.g., —CH₂—CHCH₃—) are possible, the linker ispreferably unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure —(CH₂)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

The morpholino subunits may have the structure:

where Pi is a base-pairing moiety, and the linkages depicted aboveconnect the nitrogen atom of (i) to the 5′ carbon of an adjacentsubunit. The base-pairing moieties Pi may be the same or different, andare generally designed to provide a sequence which binds to a targetnucleic acid. The use of embodiments of linkage types (b1), (b2) and(b3) above to link morpholino subunits may be illustrated graphically asfollows:

Preferably, all cationic linkages in the oligomer are of the same type;i.e. all of type (b1), all of type (b2), or all of type (b3).

In further embodiments, the cationic linkages are selected from linkages(b1′) and (b1″) as shown below, where (b1″) is referred to herein as a“Pip” linkage and (b1″) is referred to herein as a “GuX” linkage:

In the structures above, W is S or O, and is preferably O; each of R¹and R² is independently selected from hydrogen and lower alkyl, and ispreferably methyl; and A represents hydrogen or a non-interferingsubstituent on one or more carbon atoms in (b1′) and (b1″). Preferably,the ring carbons in the piperazine ring are unsubstituted; however, theymay include non-interfering substituents, such as methyl or fluorine.Preferably, at most one or two carbon atoms is so substituted. Infurther embodiments, at least 10% of the linkages are of type (b1′) or(b1″); for example, 10%-60% and preferably 20% to 50%, of the linkagesmay be of type (b1′) or (b1″).

In certain embodiments, the oligomer contains no linkages of the type(b1′) above. Alternatively, the oligomer contains no linkages of type(b1) where each R is H, R³ is H or CH₃, and R⁴ is H, CH₃, or an electronpair.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is modified with a pendant cationic group as described above.

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcould be used. For example, a 5′ nitrogen atom on a morpholino ringcould be employed in a sulfamide linkage or a urea linkage (wherephosphorus is replaced with carbon or sulfur, respectively) and modifiedin a manner analogous to the 5′-nitrogen atom in structure (b3) above.

Oligomers having any number of cationic linkages are provided, includingfully cationic-linked oligomers. Preferably, however, the oligomers arepartially charged, having, for example, 10%-80%. In preferredembodiments, about 10% to 60%, and preferably 20% to 50% of the linkagesare cationic.

In one embodiment, the cationic linkages are interspersed along thebackbone. The partially charged oligomers preferably contain at leasttwo consecutive uncharged linkages; that is, the oligomer preferablydoes not have a strictly alternating pattern along its entire length.

Also considered are oligomers having blocks of cationic linkages andblocks of uncharged linkages; for example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In one embodiment, the oligomer has approximately equal-length 5′, 3′and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, preferably greater than about70%.

Oligomers for use in for use in the methods of the invention generallyrange in length from about 10 to about 40 subunits, more preferablyabout 10 to 30 subunits, and typically 15-25 bases. For example, anoligomer of the invention having 19-20 subunits, a useful length for anantisense compound, may ideally have two to ten, e.g., four to eight,cationic linkages, and the remainder uncharged linkages. An oligomerhaving 14-15 subunits may ideally have two to seven, e.g., 3, 4, or 5,cationic linkages and the remainder uncharged linkages.

Each morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (e.g., A, G, C, T or U) or an analog, such ashypoxanthine (the base component of the nucleoside inosine) or 5-methylcytosine.

As noted above, certain embodiments are directed to oligomers comprisingnovel intersubunit linkages, including PMO-X oligomers and those havingmodified terminal groups. In some embodiments, these oligomers havehigher affinity for DNA and RNA than do the corresponding unmodifiedoligomers and demonstrate improved cell delivery, potency, and/or tissuedistribution properties compared to oligomers having other intersubunitlinkages. In one embodiment, the oligomers comprise at least oneintersubunit linkage of type (B) as defined herein. The oligomers mayalso comprise one or more intersubunit linkages of type (A) as definedherein. The structural features and properties of the various linkagetypes and oligomers are described in more detail in the followingdiscussion. The synthesis of these and related oligomers is described inco-owned U.S. application Ser. No. 13/118,298, which is incorporated byreference in its entirety.

Linkage (A)

Applicants have found that enhancement of antisense activity,biodistribution and/or other desirable properties can be optimized bypreparing oligomers having various intersubunit linkages. For example,the oligomers for use in the methods of the invention may optionallycomprise one or more intersubunit linkages of type (A), and in certainembodiments the oligomers comprise at least one linkage of type (A). Insome other embodiments each linkage of type (A) has the same structure.Linkages of type (A) may include linkages disclosed in co-owned U.S.Pat. No. 7,943,762 which is hereby incorporated by reference in itsentirety. Linkage (A) has the following structure (I), wherein 3′ and 5′indicate the point of attachment to the 3′ and 5′ ends, respectively, ofthe morpholino ring (i.e., structure (i) discussed below):

or a salt or isomer thereof, wherein:

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH₃)₂, —NR¹R², —OR³ or;

Y is, at each occurrence, independently O or —NR²,

R¹ is, at each occurrence, independently hydrogen or methyl;

R² is, at each occurrence, independently hydrogen or -LNR⁴R⁵R⁷;

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁴ is, at each occurrence, independently hydrogen, C₁-C₆ alkyl,—C(═NH)NH₂, —Z-L-NHC(═NH)NH₂ or —[C(═O)CHR′NH]_(m)H, where Z is —C(═O)—or a direct bond, R′ is a side chain of a naturally occurring amino acidor a one- or two-carbon homolog thereof, and m is 1 to 6;

R⁵ is, at each occurrence, independently hydrogen, methyl or an electronpair;

R⁶ is, at each occurrence, independently hydrogen or methyl;

R⁷ is, at each occurrence, independently hydrogen C₁-C₆ alkyl or C₁-C₆alkoxyalkyl; and

L is an optional linker up to 18 atoms in length comprising alkyl,alkoxy or alkylamino groups, or combinations thereof.

In some examples, the oligomer comprises at least one linkage of type(A). In some other embodiments, the oligomer includes at least twoconsecutive linkages of type (A). In further embodiments, at least 5% ofthe linkages in the oligomer are type (A); for example in someembodiments, 5%-95%, 10% to 90%, 10% to 50%, or 10% to 35% of thelinkages may be linkage type (A). In some specific embodiments, at leastone type (A) linkage is —N(CH₃)₂. In other embodiments, each linkage oftype (A) is —N(CH₃)₂. In other embodiments, at least one type (A)linkage is piperizin-1-yl, for example unsubstituted piperazin-1-yl(e.g., A2 or A3). In other embodiments, each linkage of type (A) ispiperizin-1-yl, for example unsubstituted piperazin-1-yl.

In some embodiments, W is, at each occurrence, independently S or O, andin certain embodiments W is O.

In some embodiments, X is, at each occurrence, independently —N(CH₃)₂,—NR¹R², —OR³. In some embodiments X is —N(CH₃)₂. In other aspects X is—NR¹R², and in other examples X is —OR³

In some embodiments, R¹ is, at each occurrence, independently hydrogenor methyl. In some embodiments, R¹ is hydrogen. In other embodiments Xis methyl.

In some embodiments, R² is, at each occurrence, hydrogen. In otherembodiments R² is, at each occurrence, -LNR⁴R⁵R⁷. In some embodiments,R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl. Inother embodiments, R³ is methyl. In yet other embodiments, R³ is ethyl.In some other embodiments, R³ is n-propyl or isopropyl. In some otherembodiments, R³ is C₄ alkyl. In other embodiments, R³ is C₅ alkyl. Insome embodiments, R³ is C₆ alkyl.

In certain embodiments, R⁴ is, at each occurrence, independentlyhydrogen. In other embodiments, R⁴ is methyl or ethyl. In yet otherembodiments, R⁴ is —C(═NH)NH₂, and in other embodiments, R⁴ is—Z-L-NHC(═NH)NH₂. In still other embodiments, R⁴ is —[C(═O)CHR′NH]_(m)H.Z is —C(═O)— in one embodiment and Z is a direct bond in anotherembodiment. R³ is a side chain of a naturally occurring amino acid. Insome embodiments R³ is a one- or two-carbon homolog of a side chain of anaturally occurring amino acid.

m is and integer from 1 to 6. m may be 1. m may be 2 m may be 3 m may be4 m may be 5 m may be 6

In some embodiments, R⁵ is, at each occurrence, independently hydrogen,methyl or an electron pair. In some embodiments, R⁵ is hydrogen. Inother embodiments, R⁵ is methyl.

In yet other embodiments, R⁵ is an electron pair.

In some embodiments, R⁶ is, at each occurrence, independently hydrogenor methyl. In some embodiments, R⁶ is hydrogen. In other embodiments, R⁶is methyl.

In other embodiments, R⁷ is, at each occurrence, independently hydrogenC₁-C₆ alkyl or C₂-C₆ alkoxyalkyl. In some embodiments R⁷ is hydrogen. Inother embodiments, R⁷ is C₁-C₆ alkyl. In yet other embodiments, R⁷ isC₂-C₆ alkoxyalkyl. In some embodiments, R⁷ is methyl. In otherembodiments, R⁷ is ethyl. In yet other embodiments, R⁷ is n-propyl orisopropyl. In some other embodiments, R⁷ is C₄ alkyl. In someembodiments, R⁷ is C₅ alkyl. In some embodiments, R⁷ is C₆ alkyl. In yetother embodiments, R⁷ is C₂ alkoxyalkyl. In some other embodiments, R⁷is C₃ alkoxyalkyl. In yet other embodiments, R⁷ is C₄ alkoxyalkyl. Insome embodiments, R⁷ is C₅ alkoxyalkyl. In other embodiments, R⁷ is C₆alkoxyalkyl.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g. —CH2-CH2-), alkoxy (e.g., —C—O—C—), andalkylamino (e.g. —CH2-NH—), with the proviso that the terminal atoms inL (e.g., those adjacent to carbonyl or nitrogen) are carbon atoms.Although branched linkages (e.g. —CH2-CHCH3-) are possible, the linkeris generally unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure (CH2)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

Oligomers having any number of linkage type (A) are provided. In someembodiments, the oligomer contains no linkages of type (A). In certainembodiments, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 percent of thelinkages are linkage (A). In selected embodiments, 10 to 80, 20 to 80,20 to 60, 20 to 50, 20 to 40, or 20 to 35 percent of the linkages arelinkage (A).

Linkage (B)

In some embodiments, the oligomers for use in the methods of theinvention comprise at least one linkage of type (B). For example theoligomers may comprise 1, 2, 3, 4, 5, 6 or more linkages of type (B).The type (B) linkages may be adjacent or may be interspersed throughoutthe oligomer. Linkage type (B) has the following structure (I):

or a salt or isomer thereof, wherein:

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR⁸R⁹ or —OR³; and

Y is, at each occurrence, independently O or —NR¹⁰,

R³ is, at each occurrence, independently hydrogen or C₁-C₆ alkyl;

R⁸ is, at each occurrence, independently hydrogen or C₂-C₁₂ alkyl;

R⁹ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂aralkyl or aryl;

R¹⁰ is, at each occurrence, independently hydrogen, C₁-C₁₂ alkyl or-LNR⁴R⁵R⁷;

wherein R⁸ and R⁹ may join to form a 5-18 membered mono or bicyclicheterocycle or R⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7 memberedheterocycle, and wherein when X is 4-piparazino, X has the followingstructure (III):

wherein:

R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl;

R is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl; and

R¹² is, at each occurrence, independently, hydrogen, C₁-C₁₂ alkyl,C₁-C₁₂ aminoalkyl, —NH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵, C₁-C₁₂ alkylcarbonyl,oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl,amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl.

In some examples, the oligomer comprises one linkage of type (B). Insome other embodiments, the oligomer comprises two linkages of type (B).In some other embodiments, the oligomer comprises three linkages of type(B). In some other embodiments, the oligomer comprises four linkages oftype (B). In still other embodiments, the linkages of type (B) areconsecutive (i.e., the type (B) linkages are adjacent to each other). Infurther embodiments, at least 5% of the linkages in the oligomer aretype (B); for example in some embodiments, 5%-95%, 10% to 90%, 10% to50%, or 10% to 35% of the linkages may be linkage type (B).

In other embodiments, R³ is, at each occurrence, independently hydrogenor C₁-C₆ alkyl. In yet other embodiments, R³ may be methyl. In someembodiments, R³ may be ethyl. In some other embodiments, R³ may ben-propyl or isopropyl. In yet other embodiments, R³ may be C₄ alkyl. Insome embodiments, R³ may be C₅ alkyl. In some embodiments, R³ may be C₆alkyl.

In some embodiments, R⁸ is, at each occurrence, independently hydrogenor C₂-C₁₂ alkyl. In some embodiments, R⁸ is hydrogen. In yet otherembodiments, R⁸ is ethyl. In some other embodiments, R⁸ is n-propyl orisopropyl. In some embodiments, R⁸ is C₄ alkyl. In yet otherembodiments, R⁸ is C₅ alkyl. In other embodiments, R⁸ is C₆ alkyl. Insome embodiments, R⁸ is C₇ alkyl. In yet other embodiments, R⁸ is C₈alkyl. In other embodiments, R⁸ is C₉ alkyl. In yet other embodiments,R⁸ is C₁₀ alkyl. In some other embodiments, R⁸ is C₁₁ alkyl. In yetother embodiments, R⁸ is C₁₂ alkyl. In some other embodiments, R⁸ isC₂-C₁₂ alkyl and the C₂-C₁₂ alkyl includes one or more double bonds(e.g., alkene), triple bonds (e.g., alkyne) or both. In someembodiments, R⁸ is unsubstituted C₂-C₁₂ alkyl.

In some embodiments, R⁹ is, at each occurrence, independently hydrogen,C₁-C₁₂ alkyl, C₁-C₁₂ aralkyl or aryl. In some embodiments, R⁹ ishydrogen. In yet other embodiments, R⁹ is C₁-C₁₂ alkyl. In otherembodiments, R⁹ is methyl. In yet other embodiments, R⁹ is ethyl. Insome other embodiments, R⁹ is n-propyl or isopropyl. In someembodiments, R⁹ is C₄ alkyl. In some embodiments, R⁹ is C₅ alkyl. In yetother embodiments, R⁹ is C₆ alkyl. In some other embodiments, R⁹ is C₇alkyl. In some embodiments, R⁹ is C₈ alkyl. In some embodiments, R⁹ isC₉ alkyl. In some other embodiments, R⁹ is C₁₀ alkyl. In some otherembodiments, R⁹ is C₁₁ alkyl. In yet other embodiments, R⁹ is C₁₂ alkyl.

In some other embodiments, R⁹ is C₁-C₁₂ aralkyl. For example, n someembodiments R⁹ is benzyl and the benzyl may be optionally substituted oneither the phenyl ring or the benzylic carbon. Substituents in thisregards include alkyl and alkoxy groups, for example methyl or methoxy.In some embodiments, the benzyl group is substituted with methyl at thebenzylic carbon. For example, in some embodiments, R⁹ has the followingstructure (XIV):

In other embodiments, R⁹ is aryl. For example, in some embodiments R⁹ isphenyl, and the phenyl may be optionally substituted. Substituents inthis regard substituents include alkyl and alkoxy groups, for examplemethyl or methoxy. In other embodiments, R⁹ is phenyl and the phenylcomprises a crown ether moiety, for example a 12-18 membered crownether. In one embodiment the crown ether is 18 membered and may furthercomprise and additional phenyl moiety. For example, in one embodiment R⁹has one of the following structures (XV) or XVI):

In some embodiments, R¹⁰ is, at each occurrence, independently hydrogen,C₁-C₁₂ alkyl or -LNR⁴R⁵R⁷, wherein R⁴, R⁵ and R⁷ are as defined abovewith respect to linkage (A). In other embodiments, R¹⁰ is hydrogen. Inother embodiments, R¹⁰ is C₁-C₁₂ alkyl, and in other embodiments R¹⁰ is-LNR⁴R⁵R⁷. In some embodiments, R¹⁰ is methyl. In yet other embodiments,R¹⁰ is ethyl. In some embodiments, R¹⁰ is C₃ alkyl. In some embodiments,R¹⁰ is C₄ alkyl. In yet other embodiments, R¹⁰ is C₅ alkyl. In someother embodiments, R¹⁰ is C₆ alkyl. In other embodiments, R¹⁰ is C₇alkyl. In yet other embodiments, R¹⁰ is C₈ alkyl. In some embodiments,R¹⁰ is C₉ alkyl. In other embodiments, R¹⁰ is C₁₀ alkyl. In yet otherembodiments, R¹⁰ is C₁₁ alkyl. In some other embodiments, R¹⁰ is C₁₂alkyl.

In some embodiments, R⁸ and R⁹ join to form a 5-18 membered mono orbicyclic heterocycle. In some embodiments the heterocycle is a 5 or 6membered monocyclic heterocycle. For example, in some embodimentslinkage (B) has the following structure (IV):

In other embodiments, heterocycle is bicyclic, for example a 12-memberedbicyclic heterocycle. The heterocycle may be piperizinyl. Theheterocycle may be morpholino. The heterocycle may be piperidinyl. Theheterocycle may be decahydroisoquinoline. Representative heterocyclesinclude the following:

In some embodiments, R¹¹ is, at each occurrence, independently C₂-C₁₂alkyl, C₁-C₁₂ aminoalkyl, aryl, heteroaryl or heterocyclyl.

In some embodiments, R¹¹ is C₂-C₁₂ alkyl. In some embodiments, R¹¹ isethyl. In other embodiments, R¹¹ is C₃ alkyl. In yet other embodiments,R¹¹ is isopropyl. In some other embodiments, R¹¹ is C₄ alkyl. In otherembodiments, R¹¹ is C₅ alkyl. In some embodiments, R¹¹ is C₆ alkyl. Inother embodiments, R¹¹ is C₇ alkyl. In some embodiments, R¹¹ is C₈alkyl. In other embodiments, R¹¹ is C₉ alkyl. In yet other embodiments,R¹¹ is C₁₀ alkyl. In some other embodiments, R¹¹ is C₁₁ alkyl. In someembodiments, R¹¹ is C₁₂ alkyl.

In other embodiments, R¹¹ is C₁-C₁₂ aminoalkyl. In some embodiments, R¹¹is methylamino. In some embodiments, R¹¹ is ethylamino. In otherembodiments, R¹¹ is C₃ aminoalkyl. In yet other embodiments, R¹¹ is C₄aminoalkyl. In some other embodiments, R¹¹ is C₅ aminoalkyl. In otherembodiments, R¹¹ is C₆ aminoalkyl. In yet other embodiments, R¹¹ is C₇aminoalkyl. In some embodiments, R¹¹ is C₈ aminoalkyl. In otherembodiments, R¹¹ is C₉ aminoalkyl. In yet other embodiments, R¹¹ is C₁₀aminoalkyl. In some other embodiments, R¹¹ is C₁₁ aminoalkyl. In otherembodiments, R¹¹ is C₁₂ aminoalkyl.

In other embodiments, R¹¹ is C₁-C₁₂ alkylcarbonyl. In yet otherembodiments, R¹¹ is C₁ alkylcarbonyl. In other embodiments, R¹¹ is C₂alkylcarbonyl. In some embodiments, R¹¹ is C₃ alkylcarbonyl. In yetother embodiments, R¹¹ is C₄ alkylcarbonyl. In some embodiments, R¹¹ isC₅ alkylcarbonyl. In some other embodiments, R¹¹ is C₆ alkylcarbonyl. Inother embodiments, R¹¹ is C₇ alkylcarbonyl. In yet other embodiments,R¹¹ is C₈ alkylcarbonyl. In some embodiments, R¹¹ is C₉ alkylcarbonyl.In yet other embodiments, R¹¹ is C₁₀ alkylcarbonyl. In some otherembodiments, R¹¹ is C₁₁ alkylcarbonyl. In some embodiments, R¹¹ is C₁₂alkylcarbonyl. In yet other embodiments, R¹¹ is —C(═O)(CH₂)_(n)CO₂H,where n is 1 to 6. For example, in some embodiments, n is 1. In otherembodiments, n is 2. In yet other embodiments, n is 3. In some otherembodiments, n is 4. In yet other embodiments, n is 5. In otherembodiments, n is 6.

In other embodiments, R¹¹ is aryl. For example, in some embodiments, R¹¹is phenyl. In some embodiments, the phenyl is substituted, for examplewith a nitro group.

In other embodiments, R¹¹ is heteroaryl. For example, in someembodiments, R¹¹ is pyridinyl. In other embodiments, R¹¹ is pyrimidinyl.

In other embodiments, R¹¹ is heterocyclyl. For example, in someembodiments, R¹¹ is piperidinyl, for example piperidin-4-yl.

In some embodiments, R¹¹ is ethyl, isopropyl, piperidinyl, pyrimidinyl,cholate, deoxycholate, or —C(═O)(CH₂)_(n)CO₂H, where n is 1 to 6.

In some embodiments, R is an electron pair. In other embodiments, R ishydrogen, and in other embodiments R is C₁-C₁₂ alkyl. In someembodiments, R is methyl. In some embodiments, R is ethyl. In otherembodiments, R is C₃ alkyl. In yet other embodiments, R is isopropyl. Insome other embodiments, R is C₄ alkyl. In yet other embodiments, R is C₅alkyl. In some embodiments, R is C₆ alkyl. In other embodiments, R is C₇alkyl. In yet other embodiments, R is C₈ alkyl. In other embodiments, Ris C₉ alkyl. In some embodiments, R is C₁₀ alkyl. In yet otherembodiments, R is C₁₁ alkyl. In some embodiments, R is C₁₂ alkyl.

In some embodiments, R¹² is, at each occurrence, independently,hydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵,oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl,amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl

In some embodiments, R¹² is hydrogen. In some embodiments, R¹² is C₁-C₁₂alkyl. In some embodiments, R¹² is C₁-C₁₂ aminoalkyl. In someembodiments, R¹² is —NH₂. In some embodiments, R¹² is —NR¹³R¹⁴. In someembodiments, R¹² is —NR¹³R¹⁴R¹⁵. In some embodiments, R¹² is C₁-C₁₂alkylcarbonyl. In some embodiments, R¹² is oxo. In some embodiments, R¹²is —CN. In some embodiments, R¹² is trifluoromethyl. In someembodiments, R¹² is amidyl. In some embodiments, R¹² is amidinyl. Insome embodiments, R¹² is amidinylalkyl. In some embodiments, R¹² isamidinylalkylcarbonyl. In some embodiments, R¹² is guanidinyl, forexample mono methylguanidynyl or dimethylguanidinyl. In someembodiments, R¹² is guanidinylalkyl. In some embodiments, R¹² isamidinylalkylcarbonyl. In some embodiments, R¹² is cholate. In someembodiments, R¹² is deoxycholate. In some embodiments, R¹² is aryl. Insome embodiments, R¹² is heteroaryl. In some embodiments, R¹² isheterocycle. In some embodiments, R¹² is —SR¹³. In some embodiments, R¹²is C₁-C₁₂ alkoxy. In some embodiments, R¹² is dimethyl amine.

In other embodiments, R¹² is methyl. In yet other embodiments, R¹² isethyl. In some embodiments, R¹² is C₃ alkyl. In some embodiments, R¹² isisopropyl. In some embodiments, R¹² is C₄ alkyl. In other embodiments,R¹² is C₅ alkyl. In yet other embodiments, R¹² is C₆ alkyl. In someother embodiments, R¹² is C₇ alkyl. In some embodiments, R¹² is C₈alkyl. In yet other embodiments, R¹² is C₉ alkyl. In some embodiments,R¹² is C₁₀ alkyl. In yet other embodiments, R¹² is C₁₁ alkyl. In otherembodiments, R¹² is C₁₂ alkyl. In yet other embodiments, the alkylmoiety is substituted with one or more oxygen atom to form an ethermoiety, for example a methoxymethyl moiety.

In some embodiments, R¹² is methylamino. In other embodiments, R¹² isethylamino. In yet other embodiments, R¹² is C₃ aminoalkyl. In someembodiments, R¹² is C₄ aminoalkyl. In yet other embodiments, R¹² is C₅aminoalkyl. In some other embodiments, R¹² is C₆ aminoalkyl. In someembodiments, R¹² is C₇ aminoalkyl. In some embodiments, R¹² is C₈aminoalkyl. In yet other embodiments, R¹² is C₉ aminoalkyl. In someother embodiments, R¹² is C₁₀ aminoalkyl. In yet other embodiments, R¹²is C₁₁ aminoalkyl. In other embodiments, R¹² is C₁₂ aminoalkyl. In someembodiments, the amino alkyl is a dimethylamino alkyl.

In yet other embodiments, R¹² is acetyl. In some other embodiments, R¹²is C₂ alkylcarbonyl. In some embodiments, R¹² is C₃ alkylcarbonyl. Inyet other embodiments, R¹² is C₄ alkylcarbonyl. In some embodiments, R¹²is C₅ alkylcarbonyl. In yet other embodiments, R¹² is C₆ alkylcarbonyl.In some other embodiments, R¹² is C₇ alkylcarbonyl. In some embodiments,R¹² is C₈ alkylcarbonyl. In yet other embodiments, R¹² is C₉alkylcarbonyl. In some other embodiments, R¹² is C₁₀ alkylcarbonyl. Insome embodiments, R¹² is C₁₁ alkylcarbonyl. In other embodiments, R¹² isC₁₂ alkylcarbonyl. The alkylcarbonyl is substituted with a carboxymoiety, for example the alkylcarbonyl is substituted to form a succinicacid moiety (i.e., a 3-carboxyalkylcarbonyl). In other embodiments, thealkylcarbonyl is substituted with a terminal —SH group.

In some embodiments, R¹² is amidyl. In some embodiments, the amidylcomprises an alkyl moiety which is further substituted, for example with—SH, carbamate, or combinations thereof. In other embodiments, theamidyl is substituted with an aryl moiety, for example phenyl. Incertain embodiments, R¹² may have the following structure (IX):

wherein R¹⁶ is, at each occurrence, independently hydrogen, C₁-C₁₂alkyl, C₁-C₁₂ alkoxy, —CN, aryl or heteroaryl.

In some embodiments, R¹² is methoxy. In other embodiments, R¹² isethoxy. In yet other embodiments, R¹² is C₃ alkoxy. In some embodiments,R¹² is C₄ alkoxy. In some embodiments, R¹² is C₅ alkoxy. In some otherembodiments, R¹² is C₆ alkoxy. In other embodiments, R¹² is C₇ alkoxy.In some other embodiments, R¹² is C₈ alkoxy. In some embodiments, R¹² isC₉ alkoxy. In other embodiments, R¹² is C₁₀ alkoxy. In some embodiments,R¹² is C₁₁ alkoxy. In yet other embodiments, R¹² is C₁₂ alkoxy.

In certain embodiments, R¹² is pyrrolidinyl, for examplepyrrolidin-1-yl. In other embodiments, R¹² is piperidinyl, for examplepiperidin-1-yl or piperidin-4-yl. In other embodiment, R¹² ismorpholino, for example morpholin-4-yl. In other embodiments, R¹² isphenyl, and in even further embodiments, the phenyl is substituted, forexample with a nitro group. In still other embodiments, R¹² ispyrimidinyl, for example pyrimidin-2-yl.

In other embodiments, R¹³, R¹⁴ and R¹⁵ are, at each occurrence,independently C₁-C₁₂ alkyl. In some embodiments, R¹³, R¹⁴ or R¹⁵ ismethyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is ethyl. In otherembodiments, R¹³, R¹⁴ or R¹⁵ is C₃ alkyl. In yet other embodiments, R¹³,R¹⁴ or R¹⁵ is isopropyl. In other embodiments, R¹³, R¹⁴ or R¹⁵ is C₄alkyl. In some embodiments, R¹³, R¹⁴ or R¹⁵ is C₅ alkyl. In some otherembodiments, R¹³, R¹⁴ or R¹⁵ is C₆ alkyl. In other embodiments, R¹³, R¹⁴or R¹⁵ is C₇ alkyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is C₈alkyl. In other embodiments, R¹³, R¹⁴ or R¹⁵ is C₉ alkyl. In someembodiments, R¹³, R¹⁴ or R¹⁵ is C₁₀ alkyl. In some embodiments, R¹³, R¹⁴or R¹⁵ is C₁₁ alkyl. In yet other embodiments, R¹³, R¹⁴ or R¹⁵ is C₁₂alkyl.

As noted above, in some embodiments, R¹² is amidyl substituted with anaryl moiety. In this regard, each occurrence of R¹⁶ may be the same ordifferent. In certain of these embodiments, R¹⁶ is hydrogen. In otherembodiments, R¹⁶ is —CN. In other embodiments, R¹⁶ is heteroaryl, forexample tetrazolyl. In certain other embodiments, R¹⁶ is methoxy. Inother embodiments, R¹⁶ is aryl, and the aryl is optionally substituted.Optional substitutents in this regard include: C₁-C₁₂ alkyl, C₁-C₁₂alkoxy, for example methoxy; trifluoromethoxy; halo, for example chloro;and trifluoromethyl.

In other embodiments, R¹⁶ is methyl. In yet other embodiments, R¹⁶ isethyl. In some embodiments, R¹⁶ is C₃ alkyl. In some other embodiments,R¹⁶ is isopropyl. In yet other embodiments, R¹⁶ is C₄ alkyl. In otherembodiments, R¹⁶ is C₅ alkyl. In yet other embodiments, R¹⁶ is C₆ alkyl.In some other embodiments, R¹⁶ is C₇ alkyl. In some embodiments, R¹⁶ isC₈ alkyl. In yet other embodiments, R¹⁶ is C₉ alkyl. In some otherembodiments, R¹⁶ is C₁₀ alkyl. In other embodiments, R¹⁶ is C₁₁ alkyl.In some other embodiments, R¹⁶ is C₁₂ alkyl.

In some embodiments, R¹⁶ is methoxy. In some embodiments, R¹⁶ is ethoxy.In yet other embodiments, R¹⁶ is C₃ alkoxy. In some other embodiments,R¹⁶ is C₄ alkoxy. In other embodiments, R¹⁶ is C₅ alkoxy. In some otherembodiments, R¹⁶ is C₆ alkoxy. In yet other embodiments, R¹⁶ is C₇alkoxy. In some other embodiments, R¹⁶ is C₈ alkoxy. In yet otherembodiments, R¹⁶ is C₉ alkoxy. In some other embodiments, R¹⁶ is C₁₀alkoxy. In some embodiments, R¹⁶ is C₁₁ alkoxy. In some otherembodiments, R¹⁶ is C₁₂ alkoxy.

In some other embodiments, R⁸ and R⁹ join to form a 12-18 membered crownether. For example, in some embodiments, the crown ether s 18 membered,and in other embodiments the crown ether is 15 membered. In certainembodiments, R⁸ and R⁹ join to form a heterocycle having one of thefollowing structures (X) or (XI):

In some embodiments, R⁸, R⁹ or R³ join with R¹⁰ to form a 5-7 memberedheterocycle. For example, in some embodiments, R³ joins with R¹⁰ to forma 5-7 membered heterocycle. In some embodiments, the heterocycle is5-membered. In other embodiments, the heterocycle is 6-membered. Inother embodiments, the heterocycle is 7-membered. In some embodiments,the heterocycle is represented by the following structure (XII):

wherein Z′ represents a 5-7 membered heterocycle. In certain embodimentsof structure (XI), R¹² is hydrogen at each occurrence. For example,linkage (B) may have one of the following structures (B1), (B2) or (B3):

In certain other embodiments, R¹² is C₁-C₁₂ alkylcarbonyl or amidylwhich is further substituted with an arylphosphoryl moiety, for examplea triphenyl phosporyl moiety. Examples of linkages having this structureinclude B56 and B55.

In certain embodiment, linkage (B) does not have any of the structuresA1-A5. Table 3 shows representative linkages of type (A) and (B).

TABLE 3 Representative Intersubunit Linkages No. Name Structure A1 PMO

A2 PMO⁺ (unprotonated form depicted)

A3 PMO⁺ (+)

A4 PMO^(mepip) (m+)

A5 PMO^(GUX)

B1 PMO^(cp)

B2 PMO^(cps)

B3 PMO^(cpr)

B4 PMO^(Shc)

B5 PMO^(morpholino) (m)

B6 PMO^(tri) (t)

B7 PMO^(hex) (h)

B8 PMO^(dodec)

B9 PMO^(dihex)

B10 PMO^(apn) (a)

B11 PMO^(pyr) (p)

B12 PMO^(pyr) (HCl Salt)

B13 PMO^(rba)

B14 PMO^(sba)

B15 PMO^(dimethylapn)

B16 PMO^(etpip)

B17 PMO^(iprpip)

B18 PMO^(pyrQMe)

B19 PMO^(cb)

B20 PMO^(ma)

B21 PMO^(bu)

B22 PMO^(bi)

B23 PMO^(pip)

B24 PMO^(odmb)

B25 PMO^(tfb)

B26 PMO^(ctfb)

B27 PMO^(ptfb)

B28 PMO^(dcb)

B29 PMO^(dmb)

B30 PMO^(hy)

B31 PMO^(6ce)

B32 PMO^(b)

B33 PMO^(q)

B34 PMO^(npp)

B35 PMO^(o)

B36 PMO^(4ce)

B37 PMO^(5ce)

B38 PMO^(f3p)

B39 PMO^(cyp)

B40 PMO^(mop)

B41 PMO^(pp)

B42 PMO^(dmepip)

B43 PMO^(NPpip)

B44 PMO^(bipip)

B45 PMO^(suc)

 46 PMO^(glutaric)

B47 PMO^(tet)

B48 PMO^(thiol) (SH)

B49 PMO^(pros)

B50 PMO^(pror)

B51 PMO^(tme)

B52 PMO^(ca)

B53 PMO^(dca)

B54 PMO^(guan) (g)

B55 PMO^(+phos)

B56 PMO^(apnphos)

In the sequences and discussion that follows, the above names for thelinkages are often used. For example, a base comprising a PMO^(apn)linkage is illustrated as ^(apn)B, where B is a base. Other linkages aredesignated similarly. In addition, abbreviated designations may be used,for example, the abbreviated designations in parentheses above may beused (e.g., ^(a)B, refers to ^(apn)B). Other readily identifiableabbreviations may also be used.

As noted above, the present methods of the invention also provide anoligomer comprising modified terminal groups. Applicants have found thatmodification of the 3′ and/or 5′ end of the oligomer with variouschemical moieties provides beneficial therapeutic properties (e.g.,enhanced cell delivery, potency, and/or tissue distribution, etc.) tothe oligomers. In various embodiments, the modified terminal groupscomprise a hydrophobic moiety, while in other embodiments the modifiedterminal groups comprise a hydrophilic moiety. The modified terminalgroups may be present with or without the linkages described above. Forexample, in some embodiments, the oligomers comprise one or moremodified terminal group and linkages of type (A), for example linkageswherein X is —N(CH₃)₂. In other embodiments, the oligomers comprise oneor more modified terminal group and linkages of type (B), for examplelinkages wherein X is 4-aminopiperidin-1-yl (i.e., APN). In yet otherembodiments, the oligomers comprise one or more modified terminal groupand a mixture of linkages (A) and (B). For example, the oligomers maycomprise one or more modified terminal group (e.g., trityl or triphenylacetyl) and linkages wherein X is —N(CH₃)₂ and linkages wherein X is4-aminopiperidin-1-yl. Other combinations of modified terminal groupsand modified linkages also provide favorable therapeutic properties tothe oligomers.

In one embodiment, the oligomers comprising terminal modifications havethe following structure (XVII):

or a salt or isomer thereof, wherein X, W and Y are as defined above forany of linkages (A) and (B) and:

R¹⁷ is, at each occurrence, independently absent, hydrogen or C₁-C₆alkyl;

R¹⁸ and R¹⁹ are, at each occurrence, independently absent, hydrogen, acell-penetrating peptide, a natural or non-natural amino acid, C₂-C₃₀alkylcarbonyl, —C(═O)OR²¹ or R²⁰;

R²⁰ is, at each occurrence, independently guanidinyl, heterocyclyl,C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl, C₇-C₃₀ aralkyl, C₃-C₃₀alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈ cycloalkylalkylcarbonyl,C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl,C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀aralkyloxycarbonyl, or —P(═O)(R²²)₂;

B is a base-pairing moiety;

L¹ is an optional linker up to 18 atoms in length comprising bondsselected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester,carbonyl, carbamate, phosphorodiamidate, phosphoroamidate,phosphorothioate, piperazine and phosphodiester; and

x is an integer of 0 or greater; and wherein at least one of R¹⁸ or R¹⁹is R²⁰; and

wherein at least one of R¹⁸ or R¹⁹ is R²⁰ and provided that both of R¹⁷and R¹⁸ are not absent.

The oligomers with modified terminal groups may comprise any number oflinkages of types (A) and (B). For example, the oligomers may compriseonly linkage type (A). For example, X in each linkage may be —N(CH₃)₂.Alternatively, the oligomers may only comprise linkage (B). In certainembodiments, the oligomers comprise a mixture of linkages (A) and (B),for example from 1 to 4 linkages of type (B) and the remainder of thelinkages being of type (A). Linkages in this regard include, but are notlimited to, linkages wherein X is aminopiperidinyl for type (B) anddimethyl amino for type (A).

In some embodiments, R¹⁷ is absent. In some embodiments, R¹⁷ ishydrogen. In some embodiments, R¹⁷ is C₁-C₆ alkyl. In some embodiments,R¹⁷ is methyl. In yet other embodiments, R¹⁷ is ethyl. In someembodiments, R¹⁷ is C₃ alkyl. In some other embodiments, R¹⁷ isisopropyl. In other embodiments, R¹⁷ is C₄ alkyl. In yet otherembodiments, R¹⁷ is C₅ alkyl. In some other embodiments, R¹⁷ is C₆alkyl.

In other embodiments, R¹⁸ is absent. In some embodiments, R¹⁸ ishydrogen. In some embodiments, R¹⁸ is a cell-penetrating peptide asdescribed in more detail below. In some embodiments, R¹⁸ is a natural ornon-natural amino acid, for example trimethylglycine. In someembodiments, R¹⁸ is R²⁰.

In other embodiments, R¹⁹ is absent. In some embodiments, R¹⁹ ishydrogen. In some embodiments, R¹⁹ is a cell-penetrating peptide asdescribed in more detail below. In some embodiments, R¹⁹ is a natural ornon-natural amino acid, for example trimethylglycine. In someembodiments, R¹⁹ is —C(═O)OR¹⁷, for example R¹⁹ may have the followingstructure:

In other embodiments R¹⁸ or R¹⁹ is C₂-C₃₀ alkylcarbonyl, for example—C(═O)(CH₂)_(n)CO₂H, where n is 1 to 6, for example 2. In otherexamples, R¹⁸ or R¹⁹ is acetyl.

In some embodiments, R²⁰ is, at each occurrence, independentlyguanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₇-C₃₀ aralkyl, C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₆-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, —C(═O)OR²¹, or —P(═O)(R²²)₂,wherein R²¹ is C₁-C₃₀ alkyl comprising one or more oxygen or hydroxylmoieties or combinations thereof and each R²² is C⁶-C¹² aryloxy.

In certain other embodiments, R¹⁹ is —C(═O)OR²¹ and R¹⁸ is hydrogen,guanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, or —P(═O)(R²²)₂, whereineach R²² is C⁶-C¹² aryloxy.

In other embodiments, R²⁰ is, at each occurrence, independentlyguanidinyl, heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl,C₃-C₃₀ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈cycloalkylalkylcarbonyl, C₇-C₃₀ arylcarbonyl, C₇-C₃₀ aralkylcarbonyl,C₂-C₃₀ alkyloxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, C₇-C₃₀aryloxycarbonyl, C₈-C₃₀ aralkyloxycarbonyl, or —P(═O)(R²²)₂. While inother examples, R²⁰ is, at each occurrence, independently guanidinyl,heterocyclyl, C₁-C₃₀ alkyl, C₃-C₈ cycloalkyl; C₆-C₃₀ aryl, C₇-C₃₀aralkyl, C₃-C₈ cycloalkylcarbonyl, C₃-C₈ cycloalkylalkylcarbonyl, C₇-C₃₀arylcarbonyl, C₇-C₃₀ aralkylcarbonyl, C₂-C₃₀ alkyloxycarbonyl, C₃-C₈cycloalkyloxycarbonyl, C₇-C₃₀ aryloxycarbonyl, C₈-C₃₀aralkyloxycarbonyl, or —P(═O)(R²²)₂.

In some embodiments R²⁰ is guanidinyl, for example mono methylguanidynylor dimethylguanidinyl. In other embodiments, R²⁰ is heterocyclyl. Forexample, in some embodiments, R²⁰ is piperidin-4-yl. In someembodiments, the piperidin-4-yl is substituted with trityl or Bocgroups. In other embodiments, R²⁰ is C₃-C₈ cycloalkyl. In otherembodiments, R²⁰ is C₆-C₃₀ aryl.

In some embodiments, R²⁰ is C₇-C₃₀ arylcarbonyl. For example, In someembodiments, R²⁰ has the following structure (XVIII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. In some embodiments,at least one R²³ is hydrogen, for example, in some embodiments, each R²³is hydrogen. In other embodiments, at least one R²³ is C₁-C₃₀ alkoxy,for example in some embodiments, each R²³ is methoxy. In otherembodiments, at least one R²³ is heteroaryl, for example in someembodiments, at least one R²³ has one of the following structures(XVIIIa) of (XVIIIb):

In still other embodiments, one R²³ joins with another R²³ to form aheterocyclyl ring. For example, in one embodiment, R²⁰ is5-carboxyfluorescein.

In other embodiments, R²⁰ is C₇-C₃₀ aralkylcarbonyl. For example, invarious embodiments, R²⁰ has one of the following structures (XIX), (XX)or (XXI):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, wherein one R²³ may joinwith another R²³ to form a heterocyclyl ring, X is —OH or halo and m isan integer from 0 to 6. In some specific embodiments, m is 0. In otherembodiments, m is 1, while in other embodiments, m is 2. In otherembodiments, at least one R²³ is hydrogen, for example in someembodiments each R²³ is hydrogen. In some embodiments, X is hydrogen. Inother embodiments, X is —OH. In other embodiments, X is Cl. In otherembodiments, at least one R²³ is C₁-C₃₀ alkoxy, for example methoxy.

In still other embodiments, R²⁰ is C₇-C₃₀ aralkyl, for example trityl.In other embodiments, R²⁰ is methoxy trityl. In some embodiments, R²⁰has the following structure (XXII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. For example, in someembodiments each R²³ is hydrogen. In other embodiments, at least one R²³is C₁-C₃₀ alkoxy, for example methoxy.

In yet other embodiments, R²⁰ is C₇-C₃₀ aralkyl and R²⁰ has thefollowing structure (XXIII):

In some embodiments, at least one R²³ is halo, for example chloro. Insome other embodiments, one R²³ is chloro in the para position.

In other embodiments, R²⁰ is C₁-C₃₀ alkyl. For example, In someembodiments, R²⁰ is a C₄-C₂₀ alkyl and optionally comprises one or moredouble bonds. For example, In some embodiments, R²⁰ is a C₄₋₁₀ alkylcomprising a triple bond, for example a terminal triple bond.

In some embodiments, R²⁰ is hexyn-6-yl. In some embodiments, R²⁰ has oneof the following structures (XXIV), (XXV), (XXVI) or (XXVII):

In still other embodiments, R²⁰ is a C₃-C₃₀ alkylcarbonyl, for example aC₃-C₁₀ alkyl carbonyl. In some embodiments, R²⁰ is —C(═O)(CH₂)_(p)SH or—C(═O)(CH₂)_(p)SSHet, wherein p is an integer from 1 to 6 and Het is aheteroaryl. For example, p may be 1 or p may be 2. In other example Hetis pyridinyl, for example pyridin-2-yl. In other embodiments, the C₃-C₃₀alkylcarbonyl is substituted with a further oligomer, for example insome embodiments the oligomer comprises a C₃-C₃₀ alkyl carbonyl at the3′ position which links the oligomer to the 3′ position of anotheroligomer. Such terminal modifications are included within the scope ofthe present disclosure.

In other embodiments, R²⁰ is a C₃-C₃₀ alkyl carbonyl which is furthersubstituted with an arylphosphoryl moiety, for example triphenylphosphoryl. Examples of such R²⁰ groups include structure 33 in Table 2.

In other examples, R₂₀ is C₃-C₈ cycloalkylcarbonyl, for example C₅-C₇alkyl carbonyl. In these embodiments, R₂₀ has the following structure(XXVIII):

wherein R²³ is, at each occurrence, independently hydrogen, halo, C₁-C₃₀alkyl, C₁-C₃₀ alkoxy, C₁-C₃₀ alkyloxycarbonyl, C₇-C₃₀ aralkyl, aryl,heteroaryl, heterocyclyl or heterocyclalkyl, and wherein one R²³ mayjoin with another R²³ to form a heterocyclyl ring. In some embodiments,R²³ is heterocyclylalkyl, for example in some embodiments R²³ has thefollowing structure:

In some other embodiments, R²⁰ is C₃-C₈ cycloalkylalkylcarbonyl. Inother embodiments, R²⁰ is C₂-C₃₀ alkyloxycarbonyl. In other embodiments,R²⁰ is C₃-C₈ cycloalkyloxycarbonyl. In other embodiments, R²⁰ is C₇-C₃₀aryloxycarbonyl. In other embodiments, R²⁰ is C₈-C₃₀ aralkyloxycarbonyl.In other embodiments, R²⁰ is —P(═O)(R²²)₂, wherein each R²² is C⁶-C¹²aryloxy, for example in some embodiments R²⁰ has the following structure(C24):

In other embodiments, R²⁰ comprises one or more halo atoms. For example,in some embodiments R²⁰ comprises a perfluoro analogue of any of theabove R²⁰ moieties. In other embodiments, R²⁰ isp-trifluoromethylphenyl, trifluoromethyltrityl, perfluoropentyl orpentafluorophenyl.

In some embodiments the 3′ terminus comprises a modification and inother embodiments the 5′ terminus comprises a modification. In otherembodiments both the 3′ and 5′ termini comprise modifications.Accordingly, in some embodiments, R¹⁸ is absent and R¹⁹ is R²⁰. In otherembodiments, R¹⁹ is absent and R¹⁸ is R²⁰. In yet other embodiments, R¹⁸and R¹⁹ are each R²⁰.

In some embodiments, the oligomer comprises a cell-penetrating peptidein addition to a 3′ or 5′ modification. Accordingly, in some embodimentsR¹⁹ is a cell-penetrating peptide and R¹⁸ is R²⁰. In other embodiments,R¹⁸ is a cell-penetrating peptide and R¹⁹ is R²⁰. In further embodimentsof the foregoing, the cell-penetrating peptide is an arginine-richpeptide.

In some embodiments, the linker ^(L) that links the 5′ terminal group(i.e., R¹⁹) to the oligomer may be present or absent. The linkercomprises any number of functional groups and lengths provided thelinker retains its ability to link the 5′ terminal group to the oligomerand provided that the linker does not interfere with the oligomer'sability to bind to a target sequence in a sequence specific manner. Inone embodiment, L comprises phosphorodiamidate and piperazine bonds. Forexample, in some embodiments L has the following structure (XXIX):

wherein R²⁴ is absent, hydrogen or C₁-C₆ alkyl. In some embodiments, R²⁴is absent. In some embodiments, R²⁴ is hydrogen. In some embodiments,R²⁴ is C₁-C₆ alkyl. In some embodiments, R²⁴ is methyl. In otherembodiments, R²⁴ is ethyl. In yet other embodiments, R²⁴ is C₃ alkyl. Insome other embodiments, R²⁴ is isopropyl. In yet other embodiments, R²⁴is C₄ alkyl. In some embodiments, R²⁴ is C₅ alkyl. In yet otherembodiments, R²⁴ is C₆ alkyl.

In yet other embodiments, R²⁰ is C₃-C₃₀ alkylcarbonyl, and R²⁰ has thefollowing structure (XXX):

wherein R²⁵ is hydrogen or —SR²⁶, wherein R²⁶ is hydrogen, C₁-C₃₀ alkyl,heterocyclyl, aryl or heteroaryl, and q is an integer from 0 to 6.

In further embodiments of any of the above, R²³ is, at each occurrence,independently hydrogen, halo, C₁-C₃₀ alkyl, C₁-C₃₀ alkoxy, aryl,heteroaryl, heterocyclyl or heterocyclalkyl.

In some other embodiments, only the 3′ terminus of the oligomer isconjugated to one of the groups noted above. In some other embodiments,only the 5′ terminus of the oligomer is conjugated to one of the groupsnoted above. In other embodiments, both the 3′ and 5′ termini compriseone of the groups noted above. The terminal group may be selected fromany one of the groups noted above or any of the specific groupsillustrated in Table 4.

TABLE 4 Representative Terminal Groups No. Name Structure C1Trimethoxybenzoyl

C2 9-fluorene-carboxyl

C3 4-carbazolylbenzoyl

C4 4-indazolylonebenzoyl

C5 Farnesyl

C6 Geranyl

C7 Prenyl

C8 Diphenylacetyl

C9 Chlorodiphenylacetyl

C10 Hydroxydiphenylacetyl

C11 Triphenylpropionyl

C12 Triphenylpropyl

C13 Triphenylacetyl

C14 Trityl (Tr)

C15 Methoxytrityl (MeOTr)

C16 Methylsuccinimidyl- cyclohexoyl

C17 Thioacetyl

C18 COCH₂CH₂SSPy

C19 Guanidinyl

C20 Trimethylglycine

C21 Lauroyl

C22 Triethyleneglycoloyl (EG3)

C23 Succinicacetyl

C24 Diphenylphosphoryl

C25 Piperidin-4-yl

C26 Tritylpiperidin-4-yl

C27 Boc-Piperidin-4-yl

C28 Hexyn-6-yl

C29 5-carboxyfluorescein

C30 Benzhydryl

C31 p-Chlorobenzhydryl

C32 Piperazinyl (pip)

C33 Triphenylphos

C34 Dimerized

Peptide Transporters

In some embodiments of the methods of the invention, the subjectoligomer is conjugated to a peptide transporter moiety, for example acell-penetrating peptide transport moiety, which is effective to enhancetransport of the oligomer into cells. For example, in some embodimentsthe peptide transporter moiety is an arginine-rich peptide. In furtherembodiments, the transport moiety is attached to either the 5′ or 3′terminus of the oligomer. When such peptide is conjugated to eithertermini, the opposite termini is then available for further conjugationto a modified terminal group as described herein.

In some embodiments of the foregoing, the peptide transport moietycomprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, andZ′ subunits,

where

(a) each X′ subunit independently represents lysine, arginine or anarginine analog, said analog being a cationic α-amino acid comprising aside chain of the structure R³³N═C(NH₂)R³⁴, where R³³ is H or R; R³⁴ isR³⁵, NH₂, NHR, or NR₃₄, where R³⁵ is lower alkyl or lower alkenyl andmay further include oxygen or nitrogen; R³³ and R³⁴ may together form aring; and the side chain is linked to said amino acid via R³³ or R³⁴;

(b) each Y′ subunit independently represents a neutral amino acid—C(O)—(CHR)_(n)—NH—, where n is 2 to 7 and each R is independently H ormethyl; and

(c) each Z′ subunit independently represents an α-amino acid having aneutral aralkyl side chain;

wherein the peptide comprises a sequence represented by one of(X′Y′X′)_(p), (X′Y′)_(m), and (X′Z′Z′), where p is 2 to 5 and m is 2 to8.

In selected embodiments, for each X′, the side chain moiety is guanidyl,as in the amino acid subunit arginine (Arg). In further embodiments,each Y′ is —CO—(CH₂)_(n)—CHR—NH—, where n is 2 to 7 and R is H. Forexample, when n is 5 and R is H, Y′ is a 6-aminohexanoic acid subunit,abbreviated herein as Ahx; when n is 2 and R is H, Y′ is a β-alaninesubunit.

In certain embodiments, peptides of this type include those comprisingarginine dimers alternating with single Y′ subunits, where Y′ is Ahx.Examples include peptides having the formula (RY′R)_(p) or the formula(RRY′)_(p), where Y′ is Ahx. In one embodiment, Y′ is a 6-aminohexanoicacid subunit, R is arginine and p is 4.

In a further embodiment, each Z′ is phenylalanine, and m is 3 or 4.

In some embodiments, the conjugated peptide is linked to a terminus ofthe oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acidsubunit and B is a β-alanine subunit.

In selected embodiments, for each X′, the side chain moiety isindependently selected from the group consisting of guanidyl(HN═C(NH₂)NH—), amidinyl (HN═C(NH₂)C—), 2-aminodihydropyrimidyl,2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl,and it is preferably selected from guanidyl and amidinyl. In oneembodiment, the side chain moiety is guanidyl, as in the amino acidsubunit arginine (Arg).

In some embodiments, the Y′ subunits are either contiguous, in that noX′subunits intervene between Y′ subunits, or interspersed singly betweenX′ subunits. However, in some embodiments the linking subunit may bebetween Y′ subunits. In one embodiment, the Y′ subunits are at aterminus of the peptide transporter; in other embodiments, they areflanked by X′ subunits. In further embodiments, each Y′ is—CO—(CH₂)_(n)—CHR—NH—, where n is 2 to 7 and R is H. For example, when nis 5 and R is H, Y′ is a 6-aminohexanoic acid subunit, abbreviatedherein as Ahx. In selected embodiments of this group, each X′ comprisesa guanidyl side chain moiety, as in an arginine subunit. Exemplarypeptides of this type include those comprising arginine dimersalternating with single Y′ subunits, where Y′ is preferably Ahx.Examples include peptides having the formula (RY′R)₄ or the formula(RRY′)₄, where Y′ is preferably Ahx. In some embodiments, the nucleicacid analog is linked to a terminal Y′ subunit, preferably at theC-terminus. In other embodiments, the linker is of the structure AhxB,where Ahx is a 6-aminohexanoic acid subunit and B is a β-alaninesubunit.

The peptide transport moieties as described above have been shown togreatly enhance cell entry of attached oligomers, relative to uptake ofthe oligomer in the absence of the attached transport moiety, andrelative to uptake by an attached transport moiety lacking thehydrophobic subunits Y′. Such enhanced uptake may be evidenced by atleast a two-fold increase, or in other embodiments a four-fold increase,in the uptake of the compound into mammalian cells relative to uptake ofthe agent by an attached transport moiety lacking the hydrophobicsubunits Y′. In some embodiments, uptake is enhanced at least twentyfold or at least forty fold, relative to the unconjugated compound.

A further benefit of the peptide transport moiety is its expectedability to stabilize a duplex between an antisense oligomer and itstarget nucleic acid sequence. While not wishing to be bound by theory,this ability to stabilize a duplex may result from the electrostaticinteraction between the positively charged transport moiety and thenegatively charged nucleic acid. In some embodiments, the number ofcharged subunits in the transporter is less than 14, as noted above, orin other embodiments between 8 and 11, since too high a number ofcharged subunits may lead to a reduction in sequence specificity.

The present disclosure also incorporates conjugates of peptide transportmoieties and nucleic acid analogues. As noted above, the peptidetransport moieties are generally effective to enhance cell penetrationof the nucleic acid analogues. The applicants have also discovered thatincluding a glycine (G) or proline (P) amino acid subunit between thenucleic acid analogue and the remainder of the peptide transport moiety(e.g., at the carboxy or amino terminus of the carrier peptide) reducesthe toxicity of the conjugate, while the efficacy remains the same or isimproved relative to conjugates with different linkages between thepeptide transport moiety and nucleic acid analogue. Thus the presentlydisclosed conjugates have a better therapeutic window and are morepromising drug candidates than other peptide-oligomer conjugates.

In addition to reduced toxicity, the presence of a glycine or prolineamino acid subunit between the nucleic acid analogue and the carrierpeptide is believed to provide additional advantages. For example,glycine is inexpensive and is easily coupled to the nucleic acidanalogue (or optional linker) without any possibility of racemization.Similarly, proline is easily coupled without racemization and alsoprovides carrier peptides which are not helix formers. Thehydrophobicity of proline may also confer certain advantages withrespect to interaction of the carrier peptide with the lipid bilayer ofcells, and carrier peptides comprising multiple prolines (for example incertain embodiments) may resist G-tetraplex formation. Finally, incertain embodiments, when the proline moiety is adjacent to an arginineamino acid subunit, the proline moiety confers metabolic stability tothe conjugates since the argine-proline amide bond is not cleavable bycommon endopeptidases.

In some embodiments, conjugation of peptides to antisenseoligonucleotides is as described in PCT publication W02012/1150960 whichis incorporated by reference in its entirety. In a particularembodiment, for example, a peptide conjugated oligonucleotide utilizesglycine as the linker between the CPP and the antisense oligonucleotide.For example, antisense oligonucleotides of the invention can be coupledto an arginine-rich peptide, such as (Arg)6Gly (6 arginine and 1 glycinelinked to an oligonucleotide). As an example, this peptide can beconjugated to a PMO and is known as “R6-G-PMO”.

Further exemplary arginine-rich cell-penetrating peptide transporterscomprising various linkers (C, G, P, Ahx, B) are given below in Table 5.As disclosed in Table 2 above, a preferred cell-penetrating peptidetransporter is SEQ ID NO: 45 conjugated to a PMO at the 3′ terminusthrough a glycine linker (R₆G). Linkage of R₆G to the 5′ terminus isalso a preferred embodiment.

TABLE 5 Arginine-Rich Cell-Penetrating Peptide TransportersNAME (DESIGNATION) SEQUENCE SEQ ID NO.^(A) rTAT RRRQRRKKR 39 TatRKKRRQRRR 40 R₉F₂ RRRRRRRRRFF 41 R₅F₂R₄ RRRRRFFRRRR 42 R₄ RRRR 43 R₅RRRRR 44 R₆ RRRRRR 45 R₇ RRRRRRR 46 R₈ RRRRRRRR 47 R₉ RRRRRRRRR 48 (RX)₈RXRXRXRXRXRXRXRX 49 (RAhxR)₄; (P007) RAhxRRAhxRRAhxRRAhxR 50(RAhxR)₅; (CP04057) RAhxRRAhxRRAhxRRAhxRRAhxR 51 (RAhxRRBR)₂; (CP06062)RAhxRRBRRAhxRRBR 52 (RAR)₄F₂ RARRARRARRARFFC 53 (RGR)₄F₂ RGRRGRRGRRGRFFC54 ^(A)Sequences assigned to SEQ ID NOs do not include the linkageportion (e.g., C, G, P. Ahx, B, AhxB where Ahx and B refer to6-aminohexanoic acid and beta-alanine, respectively).

Methods of Use and Pharmaceutical Formulations

The present invention relates to methods for treating progeroiddiseases, such as laminopathies and related diseases or conditions in asubject in need thereof by administering to the subject an antisenseoligonucleotide as described herein, or a pharmaceutical compositioncontaining the same, wherein the oligonucleotide inhibits expression ofmutant LMNA protein mRNA by modulating splicing of LMNA pre-mRNA. Incertain aspects, for example, these and related methods can be appliedto treating progeroid laminopathies in clinical settings where progerinexpression is associated with a disease such as HGPS. These and relatedembodiments can also be combined with methods of treating or reducingprogeriod laminopathies, by concurrently or sequentially carrying outthe methods of the invention with the other treatment.

The results described herein can be generalized to the aging process andrelated conditions and diseases, beyond progeroid laminopathies. This isbecause HGPS is in many respects closely connected to normal agingprocesses. HGPS continues to be recognized as a useful model of aging(Fossel, J. Pediatr Endocrinol Metab 13 Suppl 6:1477-1481, 2000). Forinstance, the connection to atherosclerosis is very strong, especiallyof the coronary arteries. In addition, alopecia in HGPS is similar tothat seen in subjects with advanced age. Further, the prime cellularfeature of HGPS, as described many years ago by Hayflick and others(Hayflick, N Engl J Med 295:1302-1308, 1976) is early cellularsenescence. The limited number of cell divisions in HGPS fibroblasts issimilar to what is seen in fibroblasts derived from elderly individuals.That was further explored by research showing similarities in the geneexpression patterns of HGPS fibroblasts and those derived from elderlypersons, distinguishing them from fibroblasts derived from youngerpersons (Ly et al., Science 287: 2486-2492, 2000).

Accordingly, it will be understood that a method for treating aprogeroid disease or related condition as described herein can includethe treatment of a progeroid laminopathy, such as HGPS, or anotherprogeroid disease or condition, an age-related condition, acardiovascular disease or condition (such as atherosclerosis), and thelike.

It will be understood that an effective in vivo treatment regimen usingthe methods of the invention may vary according to the duration, dose,frequency and route of administration of an oligonucleotide, as well asthe condition of the subject under treatment (i.e., prophylacticadministration versus administration in response to an existingcondition). Accordingly, such in vivo therapy will often requiremonitoring by tests appropriate to the particular type of disease undertreatment, and corresponding adjustments in the dose or treatmentregimen, in order to achieve an optimal therapeutic outcome.

In certain embodiments, the methods of the invention employ formulationsor compositions suitable for the therapeutic delivery of antisenseoligomers, as described herein. Hence, in certain embodiments, themethods of the present invention employ pharmaceutically acceptablecompositions that comprise a therapeutically-effective amount of one ormore of the oligomers or agents described herein, formulated togetherwith one or more pharmaceutically acceptable carriers (additives) and/ordiluents. While it is possible for an oligomer of the present inventionto be administered alone, it is preferable to administer the compound asa pharmaceutical formulation (composition).

Methods for the delivery of nucleic acid molecules are described, forexample, in Akhtar et al., 1992, Trends Cell Bio., 2:139; and DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar;Sullivan et al., PCT WO 94/02595. These and other protocols can beutilized for the delivery of virtually any nucleic acid molecule,including the isolated oligomers of the present invention.

As detailed below, the pharmaceutical compositions used in the methodsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for buccal,sublingual, and systemic absorption, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (3) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (4) intravaginally or intrarectally, for example, as a pessary,cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)nasally.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient.

Some examples of materials that can serve as pharmaceutically-acceptablecarriers include, without limitation: (1) sugars, such as lactose,glucose and sucrose; (2) starches, such as corn starch and potatostarch; (3) cellulose, and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such ascocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Additional non-limiting examples of agents suitable for formulation withthe antisense oligomers of the instant invention include: PEG conjugatednucleic acids, phospholipid conjugated nucleic acids, nucleic acidscontaining lipophilic moieties, phosphorothioates, P-glycoproteininhibitors (such as Pluronic P85) which can enhance entry of drugs intovarious tissues; biodegradable polymers, such as poly(DL-lactide-coglycolide) microspheres for sustained release deliveryafter implantation (Emerich, D F et al., 1999, Cell Transplant, 8,47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, suchas those made of polybutylcyanoacrylate, which can deliver drugs acrossthe blood brain barrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

The methods of the invention also feature the use of a compositioncomprising surface-modified liposomes containing poly (ethylene glycol)lipids (PEG-modified, branched and unbranched or combinations thereof,or long-circulating liposomes or stealth liposomes). Oligomers of theinvention can also comprise covalently attached PEG molecules of variousmolecular weights. These formulations offer a method for increasing theaccumulation of drugs in target tissues. This class of drug carriersresists opsonization and elimination by the mononuclear phagocyticsystem (MPS or RES), thereby enabling longer blood circulation times andenhanced tissue exposure for the encapsulated drug (Lasic et al. Chem.Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43,1005-1011). Such liposomes have been shown to accumulate selectively intumors, presumably by extravasation and capture in the neovascularizedtarget tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

In a further embodiment, the methods of the present invention includesoligomer compositions prepared for delivery as described in U.S. Pat.Nos. 6,692,911, 7,163,695 and 7,070,807. In this regard, in oneembodiment, the present invention provides an oligomer of the presentinvention in a composition comprising copolymers of lysine and histidine(HK) as described in U.S. Pat. Nos. 7,163,695, 7,070,807, and 6,692,911either alone or in combination with PEG (e.g., branched or unbranchedPEG or a mixture of both), in combination with PEG and a targetingmoiety or any of the foregoing in combination with a crosslinking agent.In certain embodiments, the present invention provides antisenseoligomers in compositions comprising gluconic-acid-modifiedpolyhistidine or gluconylated-polyhistidine/transferrin-polylysine. Oneskilled in the art will also recognize that amino acids with propertiessimilar to His and Lys may be substituted within the composition.

In certain methods, the oligomers described herein may contain a basicfunctional group, such as amino or alkylamino, and are, thus, capable offorming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, e.g., Berge et al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject oligomers includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In certain methods, the oligomers of the present invention may containone or more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, e.g., Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations used in the present methods of the invention include thosesuitable for oral, nasal, topical (including buccal and sublingual),rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect. Generally, out of one hundred percent, this amountwill range from about 0.1 percent to about ninety-nine percent of activeingredient, preferably from about 5 percent to about 70 percent, mostpreferably from about 10 percent to about 30 percent.

In certain embodiments, a formulation used in the methods of theinvention comprises an excipient selected from cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and an oligomerof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable an oligomer of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association an oligomer of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations used in the invention suitable for oral administration maybe in the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. An oligomer of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms used in the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient may be mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (for example, sodium starchglycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions used according to the present invention, such as dragees,capsules, pills and granules, may optionally be scored or prepared withcoatings and shells, such as enteric coatings and other coatings wellknown in the pharmaceutical-formulating art. They may also be formulatedso as to provide slow or controlled release of the active ingredienttherein using, for example, hydroxypropylmethyl cellulose in varyingproportions to provide the desired release profile, other polymermatrices, liposomes and/or microspheres. They may be formulated forrapid release, e.g., freeze-dried. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations or dosage forms for the topical or transdermaladministration of an oligomer as provided herein include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active oligomers may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required. The ointments, pastes,creams and gels may contain, in addition to an active compound of thisinvention, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to an oligomer of thepresent invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates and polyamide powder, or mixturesof these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of an oligomer of the present invention to the body. Suchdosage forms can be made by dissolving or dispersing the oligomer in theproper medium. Absorption enhancers can also be used to increase theflux of the agent across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe agent in a polymer matrix or gel, among other methods known in theart.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more oligomers of the invention in combination with oneor more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols,antioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject oligomers may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility, amongother methods known in the art. The rate of absorption of the drug thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

Injectable depot forms may be made by forming microencapsule matrices ofthe subject oligomers in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of oligomer topolymer, and the nature of the particular polymer employed, the rate ofoligomer release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations may also prepared by entrapping the drug inliposomes or microemulsions that are compatible with body tissues.

When the oligomers of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

As noted above, the formulations or preparations used in the presentinvention may be given orally, parenterally, topically, or rectally.They are typically given in forms suitable for each administrationroute. For example, they are administered in tablets or capsule form, byinjection, inhalation, eye lotion, ointment, suppository, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

Regardless of the route of administration selected, the oligomers usedin the present invention, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, may beformulated into pharmaceutically-acceptable dosage forms by conventionalmethods known to those of skill in the art. Actual dosage levels of theactive ingredients in the pharmaceutical compositions of this inventionmay be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing unacceptably toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular oligomer of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular oligomer being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularoligomer employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compound of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, oral, intravenous, intracerebroventricularand subcutaneous doses of the compounds of this invention for a patient,when used for the indicated effects, will range from about 0.0001 toabout 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain situations, dosing is oneadministration per day. In certain embodiments, dosing is one or moreadministration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to treat thedesired condition.

Antisense molecules can be administered to cells by a variety of methodsknown to those familiar to the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres, as described herein andknown in the art. In certain embodiments, microemulsification technologymay be utilized to improve bioavailability of lipophilic (waterinsoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo,S. K., et al., Drug Development and Industrial Pharmacy, 17(12),1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci 80(7),712-714, 1991). Among other benefits, microemulsification providesenhanced bioavailability by preferentially directing absorption to thelymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

In one aspect of invention, the formulations contain micelles formedfrom an oligomer as provided herein and at least one amphiphiliccarrier, in which the micelles have an average diameter of less thanabout 100 nm. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastro-intestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Examples of amphiphilic carriers include saturated and monounsaturatedpolyethyleneglycolyzed fatty acid glycerides, such as those obtainedfrom fully or partially hydrogenated various vegetable oils. Such oilsmay advantageously consist of tri-, di-, and mono-fatty acid glyceridesand di- and mono-polyethyleneglycol esters of the corresponding fattyacids, with a particularly preferred fatty acid composition includingcapric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid14-24, palmitic acid 4-14 and stearic acid 5-15%. Another useful classof amphiphilic carriers includes partially esterified sorbitan and/orsorbitol, with saturated or mono-unsaturated fatty acids (SPAN-series)or corresponding ethoxylated analogs (TWEEN-series).

Commercially available amphiphilic carriers may be particularly useful,including Gelucire-series, Labrafil, Labrasol, or Lauroglycol (allmanufactured and distributed by Gattefosse Corporation, Saint Priest,France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurate anddi-laurate, Lecithin, Polysorbate 80, etc (produced and distributed by anumber of companies in USA and worldwide).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. In certain embodiments, polymershave a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, or from about 300 daltons to about 5,000daltons. In other embodiments, the polymer is polyethyleneglycol havinga molecular weight of from about 100 to about 5,000 daltons, or having amolecular weight of from about 300 to about 5,000 daltons. In certainembodiments, the polymer is polyethyleneglycol of 750 daltons(PEG(750)). Polymers may also be defined by the number of monomerstherein; a preferred embodiment of the present invention utilizespolymers of at least about three monomers, such PEG polymers consistingof three monomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation used in the present inventioncomprises a biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter α, β. or γ, respectively.The glucose units are linked by α-1,4-glucosidic bonds. As a consequenceof the chair conformation of the sugar units, all secondary hydroxylgroups (at C-2, C-3) are located on one side of the ring, while all theprimary hydroxyl groups at C-6 are situated on the other side. As aresult, the external faces are hydrophilic, making the cyclodextrinswater-soluble. In contrast, the cavities of the cyclodextrins arehydrophobic, since they are lined by the hydrogen of atoms C-3 and C-5,and by ether-like oxygens. These matrices allow complexation with avariety of relatively hydrophobic compounds, including, for instance,steroid compounds such as 17α-estradiol (see, e.g., van Uden et al.Plant Cell Tiss. Org. Cult. 38:1-3-113 (1994)). The complexation takesplace by Van der Waals interactions and by hydrogen bond formation. Fora general review of the chemistry of cyclodextrins, see, Wenz, Agnew.Chem. Int. Ed. Engl., 33:803-822 (1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 μm.Oligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present methods uses formulations comprising liposomescontaining an oligomer of the present invention, where the liposomemembrane is formulated to provide a liposome with increased carryingcapacity. Alternatively or in addition, the compound of the presentinvention may be contained within, or adsorbed onto, the liposomebilayer of the liposome. An oligomer of the present invention may beaggregated with a lipid surfactant and carried within the liposome'sinternal space; in these cases, the liposome membrane is formulated toresist the disruptive effects of the active agent-surfactant aggregate.

According to one embodiment of the present methods, the lipid bilayer ofa liposome contains lipids derivatized with polyethylene glycol (PEG),such that the PEG chains extend from the inner surface of the lipidbilayer into the interior space encapsulated by the liposome, and extendfrom the exterior of the lipid bilayer into the surrounding environment.

Active agents contained within liposomes of the present methods are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C14 to about C20). Polymer-derivatized lipidssuch as PEG-lipids may also be utilized for micelle formation as theywill act to inhibit micelle/membrane fusion, and as the addition of apolymer to surfactant molecules decreases the CMC of the surfactant andaids in micelle formation. Preferred are surfactants with CMCs in themicromolar range; higher CMC surfactants may be utilized to preparemicelles entrapped within liposomes of the present invention.

Liposomes used according to the present methods may be prepared by anyof a variety of techniques that are known in the art. See, e.g., U.S.Pat. No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993. For example, liposomes of the presentinvention may be prepared by diffusing a lipid derivatized with ahydrophilic polymer into preformed liposomes, such as by exposingpreformed liposomes to micelles composed of lipid-grafted polymers, atlipid concentrations corresponding to the final mole percent ofderivatized lipid which is desired in the liposome. Liposomes containinga hydrophilic polymer can also be formed by homogenization, lipid-fieldhydration, or extrusion techniques, as are known in the art.

In another exemplary formulation procedure, the active agent is firstdispersed by sonication in a lysophosphatidylcholine or other low CMCsurfactant (including polymer grafted lipids) that readily solubilizeshydrophobic molecules. The resulting micellar suspension of active agentis then used to rehydrate a dried lipid sample that contains a suitablemole percent of polymer-grafted lipid, or cholesterol. The lipid andactive agent suspension is then formed into liposomes using extrusiontechniques as are known in the art, and the resulting liposomesseparated from the unencapsulated solution by standard columnseparation.

In one aspect of the present methods, the liposomes are prepared to havesubstantially homogeneous sizes in a selected size range. One effectivesizing method involves extruding an aqueous suspension of the liposomesthrough a series of polycarbonate membranes having a selected uniformpore size; the pore size of the membrane will correspond roughly withthe largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988). In certainembodiments, reagents such as DharmaFECT® and Lipofectamine® may beutilized to introduce polynucleotides or proteins into cells.

The release characteristics of a formulation used in the present methodsdepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In mostcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range istypically between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

An oligomer may be formulated to be contained within, or, adapted torelease by a surgical or medical device or implant. In certain aspects,an implant may be coated or otherwise treated with an oligomer. Forexample, hydrogels, or other polymers, such as biocompatible and/orbiodegradable polymers, may be used to coat an implant with thecompositions of the present invention (i.e., the composition may beadapted for use with a medical device by using a hydrogel or otherpolymer). Polymers and copolymers for coating medical devices with anagent are well-known in the art. Examples of implants include, but arenot limited to, stents, drug-eluting stents, sutures, prosthesis,vascular catheters, dialysis catheters, vascular grafts, prostheticheart valves, cardiac pacemakers, implantable cardioverterdefibrillators, IV needles, devices for bone setting and formation, suchas pins, screws, plates, and other devices, and artificial tissuematrices for wound healing.

In addition to the methods provided herein, the oligomers for useaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine, by analogy withother pharmaceuticals. The antisense oligomers and their correspondingformulations may be administered alone or in combination with othertherapeutic strategies in the treatment of inflammation.

In accordance with the methods of the invention, routes of antisenseoligomer delivery include, but are not limited to, various systemicroutes, including oral and parenteral routes, e.g., intravenous,subcutaneous, intraperitoneal, and intramuscular, as well as inhalation,transdermal, pulmonary and topical delivery. The appropriate route maybe determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of an antisense oligomer in the treatment of acondition of the skin may include topical delivery, while delivery of aantisense oligomer for the treatment of a respiratory condition (e.g.,COPD) may include inhalation, intranasal or pulmonary delivery. Theoligomer may also be delivered directly to the site of inflammationinfection, or to the bloodstream.

The antisense oligomer may be administered in any convenient vehiclewhich is physiologically acceptable. Such a composition may include anyof a variety of standard pharmaceutically acceptable carriers employedby those of ordinary skill in the art. Examples include, but are notlimited to, saline, phosphate buffered saline (PBS), water, aqueousethanol, emulsions, such as oil/water emulsions or triglycerideemulsions, tablets and capsules. The choice of suitable physiologicallyacceptable carrier will vary dependent upon the chosen mode ofadministration.

In some instances, as noted above, liposomes may be employed tofacilitate uptake of the antisense oligonucleotide into cells. (See,e.g., Williams, S. A., Leukemia 10(12):1980-1989, 1996; Lappalainen etal., Antiviral Res. 23:119, 1994; Uhlmann et al., antisenseoligonucleotides: a new therapeutic principle, Chemical Reviews, Volume90, No. 4, pages 544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes,Drug Carriers in Biology and Medicine, pp. 287-341, Academic Press,1979). Hydrogels may also be used as vehicles for antisense oligomeradministration, for example, as described in WO 93/01286 or PCTApplication No US1992/005305. Alternatively, the oligonucleotides may beadministered in microspheres or microparticles. (See, e.g., Wu, G. Y.and Wu, C. H., J. Biol. Chem. 262:4429-4432, 1987). Alternatively, theuse of gas-filled microbubbles complexed with the antisense oligomerscan enhance delivery to target tissues, as described in U.S. Pat. No.6,245,747.

Sustained release compositions may also be used. These may includesemipermeable polymeric matrices in the form of shaped articles such asfilms or microcapsules.

In certain embodiments, the antisense compounds may be administered inan amount and manner effective to result in a peak blood concentrationof at least 200-400 nM antisense oligomer. Typically, one or more dosesof antisense oligomer are administered, generally at regular intervals,for a period of about one to two weeks. Preferred doses for oraladministration are from about 1-100 mg oligomer per 70 kg. In somecases, doses of greater than 100 mg oligomer/patient may be necessary.For i.v. administration, preferred doses are from about 1 mg to 500 mgoligomer per 70 kg. The antisense oligomer may be administered atregular intervals for a short time period, e.g., daily for two weeks orless. However, in some cases the oligomer is administered intermittentlyover a longer period of time. Administration may be followed by, orconcurrent with, administration of an antibiotic or other therapeutictreatment. The treatment regimen may be adjusted (dose, frequency,route, etc.) as indicated, based on the results of immunoassays, otherbiochemical tests and physiological examination of the subject undertreatment.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

SEQUENCE LISTING TABLE SEQ NAME SEQUENCE ID NO: LMNA exon 11GGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTC  1GCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGC GGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCA ACTCCAGCCCCCGAACCCAGHGPS exon 11 GGCTCCCACTGCAGCAGCTCGGGGGACCCCGCTGAGTACAACCTGCGCTC  2GCGCACCGTGCTGTGCGGGACCTGCGGGCAGCCTGCCGACAAGGCATCTGCCAGCGGCTCAGGAGCCCAGGTGGGT GGACCCATCTCCTCTGGCTCTTCTGCCTCCAGTGTCACGGTCACTCGCAGCTACCGCAGTGTGGGGGGCAGTGGGGGTGGCAGCTTCGGGGACAATCTGGTCACCCGCTCCTACCTCCTGGGCA ACTCCAGCCCCCGAACCCAGExo11.25.133 CCGCTGGCAGATGCCTTGTCGGCAG  3 Exo11.25.138CTGAGCCGCTGGCAGATGCCTTGTC  4 Exo11.25.142 GCTCCTGAGCCGCTGGCAGATGCCT  5Exo11.25.145 TGGGCTCCTGAGCCGCTGGCAGATG  6 Exo11.25.149CACCTGGGCTCCTGAGCCGCTGGCA  7 Exo11.25.154 CCACCCACCTGGGCTCCTGAGCCGC  8Exo11.25.158 GGGTCCACCCACCTGGGCTCCTGAG  9 Exo11.25.162AGATGGGTCCACCCACCTGGGCTCC 10 Exo11.25.166 GAGGAGATGGGTCCACCCACCTGGG 11Exo11.25.170 GCCAGAGGAGATGGGTCCACCCACC 12 Exo11.25.174AAGAGCCAGAGGAGATGGGTCCACC 13 Exo11.25.177 CAGAAGAGCCAGAGGAGATGGGTCC 14Exo11.25.181 GAGGCAGAAGAGCCAGAGGAGATGG 15 Exo11.25.185ACTGGAGGCAGAAGAGCCAGAGGAG 16 Exo10SD.25.69 ACGTGGTGGTGATGGAGCAGGTCAT 17Exo10SD.25.73 ACTCACGTGGTGGTGATGGAGCAGG 18 Exo10SD.25.79GCTACCACTCACGTGGTGGTGATGG 19 Exo10SD.25.84 CGGCGGCTACCACTCACGTGGTGGT 20Exo10SD.25.87 CAGCGGCGGCTACCACTCACGTGGT 21 Exo10SD.25.90CCTCAGCGGCGGCTACCACTCACGT 22 Exo10SD.25.92 GGCCTCAGCGGCGGCTACCACTCAC 23Exo10SD.25.96 GCTCGGCCTCAGCGGCGGCTACCAC 24 Exo11SA.25.779CGAGTCTGGGACTGACCACTCAGGC 25 Exo11SA.25.796 AGGCTCAGGCGGGACGGCGAGTCTG 26Exo11SA.25.801 AGACAAGGCTCAGGCGGGACGGCGA 27 Exo11SA.25.805AGGGAGACAAGGCTCAGGCGGGACG 28 Exo11SA.25.809 GGGAAGGGAGACAAGGCTCAGGCGG 29Exo11SA.25.814 GCCCTGGGAAGGGAGACAAGGCTCA 30 Exo11SA.25.820GTGGGAGCCCTGGGAAGGGAGACAA 31 Exo11SA.25.828 CTGCTGCAGTGGGAGCCCTGGGAAG 32Exo11SA.25.830 AGCTGCTGCAGTGGGAGCCCTGGGA 33 Exo11SA.25.836CCCCCGAGCTGCTGCAGTGGGAGCC 34 HsEx10 GCTACCACTCACGTGGTGGTGATGG-AcR₆G 35HsEx11 GGGTCCACCCACCTGGGCTCCTGAG-AcR₆G 36 HsEx10-apn GC^(apn)TACCAC^(apn) TCACG^(apn) TGGTGG^(apn) TGATGG 37 HsEx11-apn GGG^(apn)TCCACCCACC^(apn) TGGGC^(apn) TCC^(apn) TGAG 38 rTAT RRRQRRKKR 39 TatRKKRRQRRR 40 R₉F₂ RRRRRRRRRFF 41 R₅F₂R₄ RRRRRFFRRRR 42 R₄ RRRR 43 R₅RRRRR 44 R₆ RRRRRR 45 R₇ RRRRRRR 46 R₈ RRRRRRRR 47 R₉ RRRRRRRRR 48 (RX)₈RXRXRXRXRXRXRXRX 49 (RAhxR)₄; (P007) RAhxRRAhxRRAhxRRAhxR 50 (RAhxR)₅;RAhxRRAhxRRAhxRRAhxRRAhxR 51 (CP04057) (RAhxRRBR)₂; RAhxRRBRRAhxRRBR 52(CP06062) (RAR)₄F₂ RARRARRARRARFFC 53 (RGR)₄F₂ RGRRGRRGRRGRFFC 54

EXAMPLES Example 1 Treatment of HGPS Cells Using AntisenseOligonucleotides Targeting LMNA

Two primary fibroblasts lines were used, HGPS fibroblasts (HGADFN167)and control fibroblasts (HGFDFN168). HGPS and control cells were seededinto a 24-well dish at the density of about 10,000 cells/well.Morpholino oligonucleotides targeted to exons 11 and/or 10 of Lamin-Apre-mRNA were individually introduced into cultured HGPS cells either byfree uptake or by nucleofection (Amaxa, for example). For free uptake,cells were cultured for 1-2 weeks in a medium containing either 25 μM or50 μM or 80 uM of PMO oligonucleotides. Those cells were then screenedby immunofluorescence with anti-progerin or anti-lamin A/C antibodies.The fluorescence intensities of progerin staining were quantified usingZeiss fluorescence microscope and a SPOT program. The experiments wereperformed in triplicate, and the PMOs that showed effects indown-regulating progerin were selected for further analysis. Analysisincluded quantitative RT-PCR with progerin-specific primer and Westernblotting analysis with anti-progerin antibodies.

Example 2 Immunofluorescence Staining of HGPS Cells Following Treatmentwith Antisense Oligonucleotides Targeting LMNA

Immunofluorescence Staining:

For immunofluorescence, cells were seeded onto 4-well chamber slides.After fixation in 4% paraformaldehyde/PBS at room temperature for 15min, cells were permeabilized with 0.5% Triton X-100/PBS at roomtemperature for 5 min, followed by an overnight incubation in theblocking solution at 4° C. (Blocking solution: 4% BSA/TBS). Cells werestained with mouse monoclonal anti-lamin A/C (MAB3211, Chemicon) andrabbit polyclonal anti-progerin (custom peptide antibody, Yenzm) for 3hours at room temperature on the following day. Primary antibodies weredetected with Alexa Fluor-labeled secondary antibodies (Invitrogen).Slides mounted with Vectashield mounting medium containing DAPI wereobserved with a Zeiss fluorescence microscope. Exposure times andacquisition settings were established at the beginning of each set ofexperiments and kept constant for all treatments. The results of theexperiments are set forth in FIG. 2. Several of the oligonucleotidessignificantly down-regulated progerin.

Example 3 SDS-Page and Western Blotting Analysis of HGPS Cells FollowingTreatment with Antisense Oligonucleotides Targeting LMNA

SDS-PAGE and Western Blotting analysis: Treated cells were collected,rinsed twice in PBS, and then lysed in Laemmli SDS-PAGE loading buffer.Samples were heated for 15 minutes at 95° C. and then loaded onto 10%SDS-PAGE gels. As for western blot analysis, proteins were transferredonto the nitrocellulose membranes. Membranes were blocked with 5%milk/TBST at 4° C. for overnight and incubated with primary antibodiesdiluted in 4% BSA/TBST at room temperature for 1-3 hours. After washeswith TBST, the membranes were incubated in secondary antibodies dilutedat 1:5000 in 1% milk/TBST for 1 hour at room temperature. Thechemiluminescence was detected with an ECL western blotting detectionkit (Pierce). Primary antibodies used include mouse monoclonalanti-lamin A/C (MAB3211, Chemicon), rabbit polyclonal anti-progerin(custom peptide antibody, Yenzm) and rabbit polyclonal anti-actin(Pan-actin, Cell Signaling). The results of the experiments are setforth in FIG. 3. The oligonucleotide corresponding to 699 (SEQ ID NO: 4)showed significant down-regulation of progerin in the experiment,despite it being removed from, and not overlapping with, the exon 11cryptic splice site of LMNA.

Example 4 Quantitative RT-PCR Analysis of HGPS Cells Following Treatmentwith Antisense Oligonucleotides Targeting LMNA

Quantitative RT-PCR:

Quantitative RT-PCR (qRT-PCR) experiments were performed to measure theexpression levels of progerin, lamin A and β-actin in 164 fibroblasts(p13, classical HGPS) following treatment with oligonucleotide 699 (SEQID NO: 4) and 706 (SEQ ID NO: 11). All reactions were carried out intriplicate on an Applied Biosystems 7900HT Fast Real-Time PCR Systemusing SYBR Green mix (Qiagen) according to the manufacturer'sinstructions. Reaction conditions were as follows: 1 cycles of 2 min at50° C.; 1 cycle of 15 min at 95° C.; and 40 cycles of 15 s at 95° C., 1min at 57° C., and 45 s at 72° C. The sequence for the β-actin forwardprimer is TCTTTGCAGCCACATTCCCG and reverse primer isGGCTTGCGGGTGTTAAAAGC. The sequence of the forward primer for amplifyingprogerin/lamin A is GCAACAAGTCCAATGAGGACCA. The progerin- and laminA-specific reverse primers were designed according toamplification-refractory mutation system strategy, by introducing amutation at the penultimate base to increase specificity. Theprogerin-specific primer sequence is CATGATGCTGCAGTTCTGGGGGCTCTGGAC, andthat for lamin A is CATGATGCTGCAGTTCTGGGGGCTCTGGAT. The results of theexperiments are set forth in FIG. 4.

Example 5 SDS-Page and Western Blotting Analysis of HGPS Cells FollowingTreatment with Antisense Oligonucleotides Targeting LMNA

SDS-PAGE and Western Blotting analysis: Treated cells were collected,rinsed twice in PBS, and then lysed in Laemmli SDS-PAGE loading buffer.Samples were heated for 15 minutes at 95° C. and then loaded onto 10%SDS-PAGE gels. As for western blot analysis, proteins were transferredonto the nitrocellulose membranes. Membranes were blocked with 5%milk/TBST at 4° C. for overnight and incubated with primary antibodiesdiluted in 4% BSA/TBST at room temperature for 1-3 hours. After washeswith TBST, the membranes were incubated in secondary antibodies dilutedat 1:5000 in 1% milk/TBST for 1 hour at room temperature. Thechemiluminescence was detected with an ECL western blotting detectionkit (Pierce). Primary antibodies used include mouse monoclonalanti-lamin A/C (MAB3211, Chemicon), rabbit polyclonal anti-progerin(custom peptide antibody, Yenzm) and horseradish peroxidase(HRP)-conjugated anti-actin (Sigma). The results of the experiments areset forth in FIG. 5. Oligonucleotide 699 (SEQ ID NO: 4) gave rise to adown-regulation of progerin and an up-regulation of lamin A, despite itbeing removed from, and not overlapping with, the exon 11 cryptic splicesite of LMNA.

REFERENCES

-   Cao, K., C. D. Blair, et al. (2011). “Progerin and telomere    dysfunction collaborate to trigger cellular senescence in normal    human fibroblasts.” J Clin Invest.-   Egholm, M., O. Buchardt, et al. (1993). “PNA hybridizes to    complementary oligonucleotides obeying the Watson-Crick    hydrogen-bonding rules.” Nature 365(6446): 566-8.-   Kinali, M., V. Arechavala-Gomeza, et al. (2009). “Local restoration    of dystrophin expression with the morpholino oligomer AVI-4658 in    Duchenne muscular dystrophy: a single-blind, placebo-controlled,    dose-escalation, proof-of-concept study.” Lancet Neurol 8(10):    918-28.-   Osorio, F. G., C. L. Navarro, et al. (2011). “Splicing-directed    therapy in a new mouse model of human accelerated aging.” Sci Transl    Med 3(106): 106ra107.-   Scaffidi, P. and T. Misteli (2005). “Reversal of the cellular    phenotype in the premature aging disease Hutchinson-Gilford progeria    syndrome.” Nat Med 11(4): 440-5.-   Svasti, S., T. Suwanmanee, et al. (2009). “RNA repair restores    hemoglobin expression in IVS2-654 thalassemic mice.” Proc Natl Acad    Sci USA 106(4): 1205-10.

1. A method for treating Hutchinson-Gilford Progeria Syndrome (HGPS) ina subject in need thereof comprising administering to the subject anantisense oligonucleotide, wherein the oligonucleotide modulatesaberrant splicing of a human LMNA pre-mRNA, the oligonucleotide beingcomposed of morpholino subunits and phosphorus-containing intersubunitlinkages joining a morpholino nitrogen of one subunit to a 5′-exocycliccarbon of an adjacent subunit, containing about 12-40 bases; and havinga targeting sequence comprising any one of SEQ ID NOs: 3-8, 10-18, and20-34, wherein the antisense oligonucleotide is covalently attached to acell-penetrating peptide and linker moiety of the formula -G-CPP,wherein G is the linker moiety and is selected from glycine, cysteine,proline, 6-aminohexanoic acid (Ahx), β-alanine (B), and Ahx-B, and CPPis the cell-penetrating peptide and is selected from SEQ ID NOS: 39-54.2.-16. (canceled)
 17. The method of claim 1, where the morpholinosubunits in the oligonucleotide are joined by phosphorus-containinglinkages, in accordance with the following structure:

wherein Z is S or O, X=NR¹R² or OR⁶, Y=O or NR⁷, and each said linkageis selected from: (a) uncharged linkage (a), wherein each of R¹, R², R⁶,and R⁷ is independently selected from hydrogen and lower alkyl; (b1)cationic linkage (b1), wherein X=NR¹R² and Y═O, and NR¹R² represents anoptional substituted piperazino group, such thatR¹R²=—CHRCHRN(R³)(R⁴)CHRCHR—, wherein each R⁴ is H, CH₃ or null, and R³is selected from H, lower alkyl, C(═NH)NH₂, Z-L-NHC(═NH)NH₂, and[C(O)CHR′NH]_(m)H, wherein where Z is carbonyl (C(O)) or a direct bond,L is an optional linker up to 18 atoms in length having bonds selectedfrom alkyl, alkoxy, and alkylamino, R′ is a side chain of a naturallyoccurring amino acid or a one- or two-carbon homolog thereof, and m is 1to 6; (b2) cationic linkage (b2), wherein X=NR¹R² and Y=O, R¹=H or CH₃,and R²=LNR³R⁴R⁵, wherein L, R³, and R⁴ are defined as above, and R⁵ isH, lower alkyl, or lower (alkoxy)alkyl; and (b3) cationic linkage (b3),wherein Y=NR⁷ and X=OR⁶, and R7=LNR³R⁴R⁵. wherein L, R³, and R⁴ and R⁵are defined as above, and R⁶ is H or lower alkyl; and at least one saidlinkage is selected from cationic linkages (b1), (b2), and (b3).
 18. Themethod of claim 17, where each of R¹ and R², in linkages of type (a), ismethyl.
 19. The method of claim 17, where at least one linkage is oftype (b1), where each R is H, R⁴ is H, CH₃, or an electron pair, and R³is selected from H, CH₃, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂.
 20. Themethod of claim 17, where at least one linkage is of type (b1), whereeach R is H, R⁴ is an electron pair, and R³ is selected from C(═NH)NH₂and C(O)-L-NHC(═NH)NH₂.
 21. The method of claim 17, wherein at least onelinkage is of type (b1), where each R is H, R⁴ is an electron pair, andR³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂.
 22. The method ofclaim 21, where R³ is C(O)-L-NHC(NH)NH₂, and L is a hydrocarbon havingthe structure —(CH₂)_(n)—, where n is 1 to
 12. 23. The method of claim17, where at least one linkage is of type (b1), where each R is H, andeach of R³ and R⁴ is independently H or CH₃. 24.-25. (canceled)
 26. Themethod of claim 1, where the cell-penetrating peptide is attached at itsC-terminus to the 5′ end of the oligonucleotide.
 27. The method of claim1, where the cell-penetrating peptide is attached at its C-terminus tothe 3′ end of the oligonucleotide.
 28. (canceled)
 29. A method fortreating Hutchinson-Gilford Progeria Syndrome (HGPS) in a subject inneed thereof comprising administering to the subject an antisenseoligonucleotide, wherein the oligonucleotide modulates aberrant splicingof a human LMNA pre-mRNA, the oligonucleotide comprising a backbone, thebackbone comprising a sequence of morpholino ring structures joined byintersubunit linkages, the intersubunit linkages joining a 3′-end of onemorpholino ring structure to a 5′-end of an adjacent morpholino ringstructure, wherein each morpholino ring structure is bound to abase-pairing moiety, such that the oligonucleotide can bind in asequence-specific manner to a target nucleic acid, comprising atargeting sequence comprising any one of SEQ ID NOS: 3-8, 10-18 and20-34, wherein the intersubunit linkages have the following generalstructure (I):

or a salt or isomer thereof, and wherein each of the intersubunitlinkages (I) are independently linkage (A) or linkage (B): wherein forlinkage (A): W is, at each occurrence, independently S or O; X is, ateach occurrence, independently —N(CH₃)₂, —NR¹R², —OR³ or;

Y is, at each occurrence, independently O or —NR², R¹ is, at eachoccurrence, independently hydrogen or methyl; R² is, at each occurrence,independently hydrogen or —LNR⁴R⁵R⁷; R³ is, at each occurrence,independently hydrogen or C₁-C₆ alkyl; R⁴ is, at each occurrence,independently hydrogen, methyl, —C(═NH)NH₂, —Z-L-NHC(═NH)NH₂ or—[C(O)CHR′NH]_(m)H, where Z is carbonyl (C(O)) or a direct bond, R′ is aside chain of a naturally occurring amino acid or a one- or two-carbonhomolog thereof, and m is 1 to 6; R⁵ is, at each occurrence,independently hydrogen, methyl or an electron pair; R⁶ is, at eachoccurrence, independently hydrogen or methyl; R⁷ is, at each occurrence,independently hydrogen C₁-C₆ alkyl or C₁-C₆ alkoxyalkyl; L is anoptional linker up to 18 atoms in length comprising alkyl, alkoxy oralkylamino groups, or combinations thereof; and wherein for linkage (B):W is, at each occurrence, independently S or O; X is, at eachoccurrence, independently —NR⁸R⁹ or —OR³; and Y is, at each occurrence,independently O or —NR¹⁰, R⁸ is, at each occurrence, independentlyhydrogen or C₂-C₁₂ alkyl; R⁹ is, at each occurrence, independentlyhydrogen, C₁-C₁₂ alkyl, C₁-C₁₂ aralkyl or aryl; R¹⁰ is, at eachoccurrence, independently hydrogen, C₁-C₁₂ alkyl or -LNR⁴R⁵R; wherein R⁸and R⁹ may join to form a 5-18 membered mono or bicyclic heterocycle orR⁸, R⁹ or R³ may join with R¹⁰ to form a 5-7 membered heterocycle, andwherein when X is 4-piparazino, X has the following structure (III):

wherein: R¹¹ is, at each occurrence, independently C₂-C₁₂ alkyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ alkylcarbonyl, aryl, heteroaryl or heterocyclyl; andR is, at each occurrence, independently an electron pair, hydrogen orC₁-C₁₂ alkyl; and R¹² is, at each occurrence, independently, hydrogen,C₁-C₁₂ alkyl, C₁-C₁₂ aminoalkyl, —NH₂, —NR¹³R¹⁴, —NR¹³R¹⁴R¹⁵, C₁-C₁₂alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl,amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl,guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl,heterocycle, —SR¹³ or C₁-C₁₂ alkoxy, wherein R¹³, R¹⁴ and R¹⁵ are, ateach occurrence, independently C₁-C₁₂ alkyl; and wherein the antisenseoligonucleotide is covalently attached to a cell-penetrating peptide andlinker moiety of the formula -G-CPP, wherein G is the linker moietyselected from glycine, cysteine, proline, 6-aminohexanoic acid (Ahx),β-alanine (B), and Ahx-B, and CPP is the cell-penetrating peptide and isselected from SEQ ID NOS: 39-54.
 30. The method of claim 29, where atleast 5% of the intersubunit linkages are linkage (B).
 31. The method ofclaim 29, where 10% to 50% of the intersubunit linkages are linkage (B).32. The method of claim 29, where each linkage (B) has the samestructure at each occurrence.
 33. The method of claim 29, where each Yand each W are O.
 34. The method of claim 29, where the targetingsequence comprises any one of SEQ ID NOs: 3-7 or 14-16.
 35. The methodof claim 29, where the targeting sequence consists essentially of SEQ IDNO:
 4. 36. The method of claim 29, where the targeting sequence consistsessentially of SEQ ID NO:
 11. 37. (canceled)
 38. The method of claim 1,where the targeting sequence consists essentially of SEQ ID NO:
 4. 39.The method of claim 1, where the targeting sequence consists essentiallyof SEQ ID NO: 11.